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Berberine: A Review of its Pharmacokinetics Properties and Therapeutic Potentials in Diverse Vascular Diseases

Traditional Chinese medicine plays a significant role in the treatment of various diseases and has attracted increasing attention for clinical applications. Vascular diseases affecting vasculature in the heart, cerebrovascular disease, atherosclerosis, and diabetic complications have compromised quality of life for affected individuals and increase the burden on health care services. Berberine, a naturally occurring isoquinoline alkaloid form Rhizoma coptidis, is widely used in China as a folk medicine for its antibacterial and anti-inflammatory properties. Promisingly, an increasing number of studies have identified several cellular and molecular targets for berberine, indicating its potential as an alternative therapeutic strategy for vascular diseases, as well as providing novel evidence that supports the therapeutic potential of berberine to combat vascular diseases. The purpose of this review is to comprehensively and systematically describe the evidence for berberine as a therapeutic agent in vascular diseases, including its pharmacological effects, molecular mechanisms, and pharmacokinetics. According to data published so far, berberine shows remarkable anti-inflammatory, antioxidant, antiapoptotic, and antiautophagic activity via the regulation of multiple signaling pathways, including AMP-activated protein kinase (AMPK), nuclear factor κB (NF-κB), mitogen-activated protein kinase silent information regulator 1 (SIRT-1), hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), janus kinase 2 (JAK-2), Ca2+ channels, and endoplasmic reticulum stress. Moreover, we discuss the existing limitations of berberine in the treatment of vascular diseases, and give corresponding measures. In addition, we propose some research perspectives and challenges, and provide a solid evidence base from which further studies can excavate novel effective drugs from Chinese medicine monomers.


INTRODUCTION
The global health burden of vascular diseases, such as atherosclerosis, cerebrovascular disease, hypertension, and complications of diabetes, is rapidly increasing (Al Rifai et al., 2021;Riccardi et al., 2021). Epidemiological surveys have shown that the increasing cost of vascular diseases worldwide compromises quality of life for individuals (Liss et al., 2021). In addition, a broad variety of factors, including inflammation, vascular dysplasia, oxidative stress, and abnormal lipid metabolism, cause vascular diseases (Guzik and Touyz, 2017;Feng et al., 2020). Hence, strategies aiming to reduce inflammation and oxidative stress and normalize the lipid metabolism are generally used to treat and prevent the vascular diseases, and statins, nonsteroidal anti-inflammatory drugs, and novel biological agents are common therapeutic agents (Oesterle et al., 2017;Lu et al., 2018;Doña et al., 2020). However, the high cost and side effect profiles of these drugs make finding cheaper alternatives with fewer side effects and similar or better therapeutic outcomes a matter of urgency. Therapies used in traditional Chinese medicines (TCM) have long been used as complementary and alternative medicines for the treatment of vascular disease in China (Cheng et al., 2017;Li et al., 2018b). Recently, these have garnered research interest owing to fewer adverse reactions and lower toxicities of these compounds compared with those identified and used in western medicine Oduro et al., 2020;Atanasov et al., 2021). Undeniably, TCM has made an indelible contribution to human health and is considered a potential source of therapies derived from natural, rather than synthetic, sources. Therefore, there is an increased emphasis on the use of medicinal plants such as those used in TCM in the development of novel drugs.
Berberine (C 20 H 18 NO 4 + , CAS no: 2086-83-1, Figure 1), a naturally occurring benzylisoquinoline alkaloid, has a long history of medical applications in TCM . As a natural bioactive ingredient, berberine mainly exists naturally in the roots, rhizomes, and stem bark of various medicinal plants from the Ranunculaceae , Rutaceae (Ryuk et al., 2012), and Berberidaceae families (Gawel et al., 2020). Berberine was reportedly used in China as a folk medicine by Shennong at approximately 3000 BC, and the first recorded use of berberine is described in the ancient Chinese medical book The Divine Farmer's Herb-Root Classic (Neag et al., 2018). The hydrochloride salt of berberine, listed as an oral antibacterial agent in Pharmacopoeia of the People's Republic of China, is a common over-the-counter medication; dosage is usually 0.1 g in pill form taken 1-3 times per day for gastrointestinal infections (Zhang et al., 2021b). Colloquially, it is known as Huangliansu (Chinese: 黄连素; literally translated into English: "the essence of Chinese goldthread"). Modern pharmacological studies have confirmed that berberine exhibits various clinically useful biological properties, including anticardiovascular disease and anticancer properties (Feng et al., 2019;Hu et al., 2019). A growing body of evidence has shown that berberine has poor bioavailability due to first-pass effects in the intestinal lumen, leading to limitations in its clinical application (Xu et al., 2019;Habtemariam, 2020b). However, berberine is currently being evaluated in clinical trials for its important clinical benefits, lower toxicity and side effects compared with currently available therapies in western medicine, with its active metabolites exerting similar bioactive properties as berberine itself (Kumar et al., 2015;Imenshahidi and Hosseinzadeh, 2019). Further studies on berberine's mechanism of action as well as new applications and novel formulations are therefore warranted.
In recent years, novel unique molecular entities derived from herbal medicines containing berberine have led to increased attention to the potential of this compound in the treatment of vascular diseases (Lee et al., 2018;Ren et al., 2019;Rajabi et al., 2021). Along with advances in pharmacological research, berberine was considered one of the most promising naturally derived drugs for the treatment of numerous human vascular diseases through the modulation of multiple signaling pathways. However, no systematic reviews on the pharmacological and pharmacokinetic properties of berberine in the context of vascular disease have been published. Therefore, in this review, we screened articles on berberine treatment in vascular diseases published in the years 2010-2021 using Web of Science, ScienceDirect, PubMed, Google Scholar and China National Knowledge Infrastructure online databases and summarized the findings to provide insights into the potential application of berberine in vascular diseases. the A ring forming a methylenedioxy group responsible for most of the biological activities of berberine, such as anticancer activity (Leyva-Peralta et al., 2019). The "C" ring contains a quaternary ammonium structure (with N + in the aromatic ring), which is necessary for the antibacterial activity (Gaba et al., 2021). In the "D" ring, C 9 and C 10 are each attached to a methoxy group. At present, structural modification studies of berberine mainly focus on the "C" and "D" rings Habtemariam 2020a); available evidence suggests that alkylation or acylation in the "D" ring resulted in hypoglycemic activity (Cheng et al., 2010;Shan et al., 2013). The introduction of cinnamic acid at 9-O position exerted strong hypoglycemic effects (Zhang et al., 2016b). C 8 and C 13 alkylation were shown to enhance cytotoxicity . Similarly, positions N 7 and C 13 are prone to modifications that enhance anticellular proliferative activity of berberine (Gaba et al., 2021). Moreover, berberine is fluorescent, with a maximum absorption wavelength of 350 nm and an emission wavelength of 530 nm in 0.01 mol/L sodium dodecyl sulfate solution. Thus, liquid chromatography-mass spectrometry and isotope labeling can be used to measure the content of berberine as part of a TCM or drugs (Chang et al., 2016;Ai et al., 2019).

PHARMACOKINETICS OF BERBERINE Absorption
Berberine exerts superior therapeutical effects on the vascular diseases, such as atherosclerosis; however, its effects are limited in clinic due to poor oral absorption and low bioavailability (Han et al., 2021b). Previous research has reported that the absolute bioavailability of berberine is 0.37% when administered in a single oral administration (48.2, 120, or 240 mg/kg body weight) in rats (Feng et al., 2021). Oral treatment with 100 mg/kg berberine has an absolute bioavailability of 0.68% as measured in rat plasma samples, with a mean maximum plasma concentration (C max ) of 9.48 ng/ml and an area under the curve (AUC) 0-36 h of 46.5 ng h/ ml (Chen et al., 2011). Sahibzada et al. (2021) found that after a single oral dose of 50 mg/kg berberine in rabbits, the C max was 0.411 μg/ml. Some studies have also reported the absorption of berberine in humans. In one study, the mean C max at 8 h post-administration was reported to be approximately 0.4 ng/ml for 400 mg berberine administered orally (n 20) (Hua et al., 2007). Another study reported that the C max of berberine in 10 healthy individuals given 500 mg berberine orally was extremely low, at 0.07 nM (Spinozzi et al., 2014). It is thought that the lower in vivo bioavailability of berberine is closely related to extensive intestinal first-pass elimination, in which the drug is filtered out of the circulation by the liver resulting in a low level of systemic circulation (Guan et al., 2018). After orally administrated with 100 mg/kg berberine to rats, approximately half of berberine ran intact through the gastrointestinal tract and another half was disposed of by the small intestine, resulting in an extremely low extent of absolute oral bioavailability (0.36%) (Liu et al., 2010b). Additionally, a caco-2 cell monolayer model was used to confirm that berberine is the substrate for the drug transporter P glycoprotein, which may contribute to the lower absorption of berberine in small intestinal epithelial cells by passive diffusion Cui et al., 2015).
With its remarkable pharmacological activity, berberine has been used for a variety of diseases in the clinic. However, due to its low in vivo bioavailability, exploring methods that increase the concentration of berberine in blood is key to improving its usefulness in this context. Although intravenous administration provides a direct approach that may improve the bioavailability of berberine, this can lead to serious side effects including respiratory arrest (Han et al., 2021b). Therefore, berberine is often administered orally in clinic. Conversion of biological small molecules into salt compounds may be a method to improve its bioavailability in vivo. The bioavailability of berberine organic acid salts, especially berberine fumarate and berberine succinate, is higher than that of berberine hydrochloride (Cui et al., 2018). Moreover, chemical structure modification can be used to improve bioavailability of this drug. Long-chain alkylation (C 5 -C 9 ) may enhance hydrophobicity, which has been shown to improve bioavailability; for example, 9-O-benzylation further enhances lipophilicity and imparts neuroprotective effect Singh et al., 2021).

Distribution
It has been demonstrated that berberine is rapidly distributed through tissues in the liver, kidneys, muscle, lungs, brain, heart, pancreas, and fat, in descending order of amount, while the concentration of berberine in most of these tissues was higher than that in plasma 4 h after oral administration at a dose of 200 mg/kg in rats. Moreover, berberine concentrations remained relatively stable in liver, heart, brain, muscle, and pancreas tissue in rats .
However, recent studies on the distribution of berberine in vivo are rare, which may be attributed to the broad tissue distribution in vivo after oral administration. The availability of new technologies such as component analysis by highperformance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESIMS)/mass spectrometry (MS) and MS imaging may permit improved exploration of the berberine tissue distribution (Jove et al., 2019). The fact that berberine is widely distributed in tissues may be useful in the treatment of some diseases, which may broaden the scope of its clinical application. For example, with the character of enrichment in the liver, oral treatment with 100 mg/kg berberine may promote the excretion of cholesterol from the liver to the bile (Li et al., 2015b). Thus, distribution of berberine may be an important pharmacokinetic property requiring further study in future.

Metabolism
One study used a sensitive HPLC-ESIMS/MS method to identify the metabolites of berberine in human plasma, of which berberrubine was most abundant, with high lipid solubility in individuals who received 15 mg/kg oral berberine chloride per day for 3 months (Spinozzi et al., 2014). Evidence showed that berberine had a similar metabolic profile in rats (100 mg/kg administered orally) and humans (300 mg administered orally three times a day for 2 days) via the urine (Qiu et al., 2008). Using Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 liquid chromatography coupled with ion trap time-of-flight mass spectrometry, Ma et al. (2013) revealed that 16 separate metabolites could be identified in rat bile, urine, and feces samples after oral administration of berberine (200 mg/kg). After a single oral administration (48.2, 120, or 240 mg/kg) of berberine in rats, the levels of phase 2 metabolites were much higher than those of phase 1 metabolites for the AUC 0-48 h values. Simultaneously, nine major metabolites of berberine (demethyleneberberine, jatrorrhizine-3-O-β-D-glucuronide, jatrorrhizine, berberrubine-9-O-β-D-glucuronide, jatrorrhizine-3-O-sulfate, berberrubine, thalfendine-10-O-β-D-glucuronide, demethyleneberberine-2-O-sulfate, and demethyleneberberine-2-O-β-D-glucuronide) were detected in rat serum using a LC-MS/MS method (Feng et al., 2021). Additionally, it was demonstrated that the metabolism of berberine by oral is closely related to liver function and gut microbiota. After oral administration of 300 mg/kg berberine in mice, cytochrome P3A11 (CYP3A11) and CYP3A25 mRNA and CYP3A11 and CYP2D22 enzyme activity levels were all found to be decreased, while the level of CYP1A2 mRNA was increased . Similarly, on oral administration of 200 mg/kg berberine in rats, the drug was shown to be metabolized in the liver by the CYP450 isoenzyme via oxidative demethylation at C 2 , C 3 , C 9, and C 10 , followed by conjugation of the hydroxyl groups with glucuronic acid . Furthermore, gut microbiota can also affect the metabolism of berberine after oral administration. It was demonstrated that 200 mg/kg berberine administered orally could be converted into absorbable dihydroberberine by nitroreductases produced by gut microbiota, which showed a nearly 5-fold higher intestinal absorption rate than berberine in rats; the dihydroberberine is then oxidized back to berberine after absorption into the intestinal tissue, and enters the blood (Feng et al., 2015;Han et al., 2021a). Also, gut microbiota was shown to convert berberine into oxyberberine through an oxidation reaction in vitro and in vivo, which exerted a much stronger binding interaction with hemoglobin than plasma (Li et al., 2020a;Chen et al., 2021).
To summarize this section, the liver and intestine are the main metabolizing organs of berberine by oral administration. Inhibiting the first-pass effect may reduce the metabolism of berberine and improve its bioavailability. Interestingly, according to an in-depth study on the metabolism of berberine in vivo, it found that phase II metabolites are the major metabolic products of berberine (Feng et al., 2021), whereas the opposite was true in previous studies . In addition, particular attention should be paid to nitroreductases produced by gut microbiota, and berberine metabolism in general, in future studies, in order to fully establish the pharmacodynamic basis of this TCM.

Excretion
To better understand the poor absorption of berberine in vivo, some researchers have paid more attention to the excretion of berberine via the digestive tract. Berberine was found in feces with a recovery rate of 22.74% after a single oral dose (200 mg/kg) in 48 h, and thalifendine was the most abundant berberine metabolite excreted in the bile, urine, and feces in rats . In another study, 18.6% of the berberine was excreted in feces as berberrubine after intragastric administration at a single dose of 48.2 mg/kg. The total recovery of berberine and its metabolites from the urine, bile, and feces was 41.2% in rats (Feng et al., 2021). To summarize, berberine and its metabolites are mainly excreted by the kidneys (urine and feces) and bile in rats and mice .

EFFECTS OF BERBERINE ON VASCULAR DISEASES
Recently, vascular protective effects of berberine have been reported in experimental studies of diverse vascular diseases. Berberine has shown promising vascular protection against atherosclerosis, cerebrovascular disease, hypertension, diabetes mellitus (DM), and intestinal vascular diseases. The pharmacological properties and molecular pathways of berberine are presented in Figure 2.

Vasculature in Heart
Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide . Studies indicate that the abnormal proliferation of vascular smooth muscle cells (VSMCs) is involved in the pathogenesis of CVD (Xiang et al., 2020;Zhang et al., 2021a). It has also been demonstrated that berberine (10, 30 and 100 μmol/L) could inhibit angiotensin IV-induced proliferation in cultured VSMCs by targeting the peroxisome proliferator-activated receptor α (PPAR-α)-nitric oxide (NO) signaling pathway (Qiu et al., 2017).

Myocardial Ischemia
Inflammation is one of the most observed cardiovascular conditions, and has a significant role in the progression of CVD (Golia et al., 2014;Lockshin et al., 2018). Aggravating inflammation may induce vascular remodeling after myocardial ischemia (MI), contributing to reduction of the ejection fraction and subsequent heart failure (Lu et al., 2015;Chong et al., 2021). It has been suggested that 50 mg/kg berberine may improve vascular inflammation and remodeling by inhibiting p38 mitogen-activated protein kinase (MAPK) activation, and activating transcription factor 2 phosphorylation (p-ATF-2) and matrix metalloprotease 2 (MMP-2) expression in rats (Li et al., 2015a). Protein hyperacetylation is associated with the development of MI (Treviño-Saldaña and García-Rivas, 2017; Aggarwal et al., 2020). Accumulating studies have demonstrated that silent information regulator 1 (SIRT-1) can regulate oxidative stress and inflammation to inhibit the development and progression of cardiac dysfunction in myocardial ischemia/reperfusion (MI/R) injury Chang et al., 2021). Owing to its strong antioxidative and anti-inflammatory activities, oral administration of berberine (200 mg/kg) conferred cardioprotective effects in rats by improving post-MI/R cardiac function recovery and reducing infarct size after MI/R injury; the mechanism of action was found to be associated with the regulation of the SIRT-1 signaling pathway .
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 As a selective barrier between tissue and blood, endothelial cells play a potential role in the control of inflammatory responses and homeostasis (Krüger-Genge et al., 2019). Endothelial cell dysfunction and/or injury can disrupt the integrity of the endothelial lining and subsequently lead to vascular disease, such as MI (Monteiro et al., 2019). Additionally, large experimental studies suggest that excessive inflammation can directly lead to endothelial cell apoptosis (Bravo-San Pedro et al., 2017;Henning et al., 2018). Lipopolysaccharide (LPS)induced inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs) were found to be inhibited by pretreatment with 5 μM berberine, mediated by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation, and increased myeloid cell leukemia 1 (MCL-1) expression and superoxide dismutase (SOD) activity . Angiogenesis, the formation of new blood vessels from preexisting ones, is indispensable for revascularization and cardiac remodeling following MI (Mathiyalagan et al., 2019;Chong et al., 2021;Tang et al., 2021). Ischemic heart disease is a leading cause of mortality and results from vascular cavity stenosis and occlusion (Garry et al., 2021). Rehabilitation of the myocardial ischemic region involves the activation of several stimulatory and inhibitory modulators of angiogenesis; the most notable of which are vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and thrombospondin 1 (TSP-1) (Detillieux et al., 2003;Frangogiannis et al., 2005;Martinez et al., 2018;Garikipati et al., 2019). Treatment with 10 mg/kg of berberine-rich extract (5 days a week by gavage) remarkably reduced heart infarct size, and increased the expression of angiogenesis-promoting factors in rats with MI/R injury, including VEGF, FGF-2 and TSP-1 (Banaei et al., 2020). In addition, microRNA plays a key role in many cardiac pathological processes, including MI (Parikh et al., 2020). Treatment with berberine in mice with MI injury was shown to lead to elevated miR-29b can activate the protein kinase B (Akt) signaling pathway, thus promoting angiogenesis and cell proliferation and migration to improve heart function (Zhu et al., 2017). A study in zebrafish embryos revealed that the level of VEGF-aa mRNA was up-regulated by berberine, which interfered with the angiogenic process, promoting bradycardia and reducing FIGURE 2 | Biological activities and potential pathways of berberine on vascular diseases. Abbreviations: Akt, protein kinase B; AMPK, AMP-activated protein kinase; AP-1, activator protein 1; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; BMPR-2, bone morphogenetic protein type 2; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HIF-1α, hypoxia-inducible factor 1α; JAK-2, janus kinase 2; MAPK, mitogen-activated protein kinase; MMP-2, matrix metalloprotease 2; NF-κB, nuclear factor κB; PI3K, phosphoinositide 3-kinase; SIRT-1, silent information regulator 1; STAT-3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor β; TRPV-4, transient receptor potential vanilloid 4; VEGF, vascular endothelial growth factor; VSMCs, vascular smooth muscle cells.
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 the cardiac output, atrial shortening fraction percentage, and atrial stroke volume (Martini et al., 2020). A complex hemodynamic pathological phenomenon exists in ischemia and reperfusion injury that can engage the metabolic and inflammatory machinery in the development of various disorders, including heart failure (Raza et al., 2020). Interestingly, intragastric administration of 100 mg/kg berberine daily for 14 days attenuated ischemia-reperfusion injury via hemodynamic improvements and inhibition of AMPK activity in both non-ischemic and ischemic areas of rat heart tissue (Chang et al., 2012). Recent evidence has confirmed that endoplasmic reticulum (ER) stress is correlated with the development and progression of various heart diseases including cardiac hypertrophy, ischemic heart diseases, and heart failure . Prolonged ER stress, however, can become a leading cause of vascular endothelial cell dysfunction and apoptosis in CVD Fatima et al., 2021). Oral administration of 200 mg/kg berberine daily for 2 weeks was reported to protect the heart from MI/R injury in rats by activating the janus kinase 2 (JAK-2)/signal transducer and activator of transcription 3 (STAT-3) signaling pathway, as well as by attenuating ER stress-induced apoptosis . Alternatively, apoptosis and inflammation are correlated with anoxiareoxygenation injury in CVD, which typically occurs during MI Gan et al., 2020). The decreased inflammatory cytokines and myocardial cell apoptosis resulting from berberine administration may alleviate anoxiareoxygenation injury by downregulating p38 MAPK-mediated nuclear factor κB (NF-κB) signaling pathway (Zhao et al., 2019c). In conclusion, given its robust anti-inflammatory, antioxidative stress, antiapoptotic, and anti-ER stress effects, berberine may effectively improve CDV and MI by inhibiting the MAPK, AMPK and NF-κB pathways and activating PPARα-NO, VEGF, and JAK-2/STAT-3 pathways ( Figure 3 and Table 1).
also reduced cholesteryl ester gathering in the aortic arch, resulting in ameliorated arterial plaque build-up via altered AMPK and NF-κB gene expression, and interrupted crosstalk between adipocytes and macrophages . Tumor necrosis factor α (TNF-α) is a major proinflammatory factor in the development of vascular inflammation (Jang et al., 2017). Aberrant inflammatory responses may result in the ablation of macrophages, aggravation of vascular endothelial injury, and abnormal tissue proliferation in atherosclerosis (Hui et al., 2010;Brown et al., 2017). In vitro studies have indicated that TNF-α-induced inflammation, which causes excessive expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) could be decreased by berberine in human aortic endothelial cells; this may be associated with inhibition of the NF-κB and AMPK pathways (Liu et al., 2015b). Oxidized LDL (ox-LDL) can act as an antigen to activate the immune inflammatory response, increasing the infiltration of inflammatory cells and the secretion of inflammatory factors in atherosclerosis (Lundberg et al., 2021). Ox-LDL-induced HUVEC proliferation and inflammatory responses were reversed with berberine, which lowered the expression of proliferating cell nuclear antigen (PCNA), NF-lB, and lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and inhibited the phosphoinositide 3-kinase (PI3K)/Akt, extracellular signalregulated kinase 1/2 (ERK-1/2), and p38 MAPK pathways (Xu et al., 2017). Ox-LDL has also been shown to injure endothelial cells directly, and contributes to endothelial dysfunction via overexpression of LOX-1, which induces a further rise in intracellular reactive oxygen species (ROS) (Kattoor et al., 2019;Akhmedov et al., 2021). Orally administrated at 156 mg/kg, berberine improved endothelial dysfunction by reducing aortic ROS generation and the release of inflammatory cytokines in the serum of a mouse model of atherosclerosis (Tan et al., 2020). Additionally, platelet-endothelial cell interactions potentiated by oxidative stress are thought to contribute to early atherosclerosis (Brown et al., 2021). Existing inflammatory and oxidative suppression of berberine prevented the development of the atherosclerotic plaque area by inhibiting translocation of NF-κB to the nucleus (Feng et al., 2017). Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane protein that belongs to the UCP family and plays an important role in lowering mitochondrial membrane potential and dissipating metabolic energy, preventing the accumulation of oxidative stress (Pierelli et al., 2017). Amazingly, treatment with 1 mmol/L berberine in drinking water led to suppression of oxidative stress and vascular inflammation by stimulating AMPKdependent UCP-2 expression in mice with atherosclerosis (Wang et al., 2011). Moreover, recent studies found that the gut microbiota played a crucial role in atherosclerosis (Jonsson and Bäckhed, 2017;Verhaar et al., 2020). Another study emphasized that the modulation of gut microbiota, specifically the abundance of the Akkermansia bacterial genus, contributed to the antiatherosclerotic and metabolic protective effects of berberine by suppressing intestinal inflammation and promoting intestinal epithelial barrier integrity (Zhu et al., 2018).
Prolonged activation of the ER stress pathway can lead to aggravated oxidative stress and endothelial cell apoptosis (Tabas, 2010;Linton et al., 2016). Kawasaki disease (KD) is an acute febrile illness characterized by systemic vasculitis, especially in coronary arteries (de Ferranti et al., 2018). Berberine exerted its protective effects on KD-induced apoptosis of human coronary artery endothelial cells by inhibiting oxidative and ER stress . Endothelial cell apoptosis induced by ER stress is closely linked with plaque progression, which can contribute to unstable atherosclerotic plaques, perhaps in response to thrombosis in atherosclerosis . Homocysteine increases damage to vascular endothelial cells, thereby reducing vasodilation factors released by endothelial cells and impairing vasodilation in the endothelium, resulting in vascular endothelial apoptosis and inducing oxidation (Salvio et al., 2021). Nevertheless, an in vivo experiment has shown that berberine increased the stability of atherosclerotic plaques and mitigated detrimental effects of vascular endothelial cell activity experimentally induced by 50 mg/kg homocysteine thiolactone, and similar results are found in vitro study induced by 1 mM homocysteine thiolactone (Li et al., 2016a). Collectively, the studies presented here ( Table 2) indicate that berberine may improve vascular endothelial damage, abnormal lipid metabolism, chronic inflammation, plaque formation, and cell apoptosis, thereby alleviating atherosclerosis ( Figure 4).

Alzheimer's Disease
Alzheimer's disease (AD) is a complex, aging-related, neurodegenerative disease, and the pathology process mainly involves β-amyloid (Aβ) overproduction and accumulation, tau hyperphosphorylation, and neuronal loss (Lian et al., 2016;Spangenberg et al., 2016;Gao et al., 2018). Administration of 0.5 μM berberine significantly suppressed Aβ-induced production of interleukin 6 (IL-6) and MCP-1 by inhibiting the activation of NF-κB and blocking the PI3K/Akt and MAPK pathways (Jia et al., 2012). Accumulated Aβ can result from abnormal processing of amyloid precursor protein (APP) in AD (O'Brien and Wong, 2011). APP C-terminal fragment levels and APP and tau hyperphosphorylation were decreased with 100 mg/kg berberine, administered by oral gavage for 4 months, via the Akt/glycogen synthase kinase 3 (GSK-3) pathway in rats with AD (Durairajan et al., 2012). Similarly, berberine modulates Aβ generation through activation of the AMPK pathway in N2a mouse neuroblastoma cells . Moreover, elevated Aβ can mediate synaptic loss and dysfunction, another pathological hallmark of AD, by targeting mitochondria (Hong et al., 2016). Pretreatment with 1 μM berberine alleviated axonal mitochondrial abnormalities by preserving the mitochondrial membrane potential and preventing decreases in ATP, increasing axonal mitochondrial density and length, and improving mitochondrial motility and trafficking in 0.5 μM Aβ cultured hippocampal mouse neurons (Zhao et al., 2019a). Additionally, neuronal loss and cerebral blood flow contributed to dysfunction in memory and reward systems in AD (Dai et al., 2009;Nobili et al., 2017). Studies found that drinking water containing 100 mg/L berberine exerted a strong neuroprotective effect, improving cognitive deficits, inhibiting the apoptosis of neurons, and promoting Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 the formation of micro-vessels by restoring cerebral blood flow and reducing Aβ accumulation in an APP/tau/PS-1 mouse model of AD (Ye et al., 2021). As shown in Table 3, the potential effects of berberine observed in the study suggest that it may effectively prevent AD by reducing accumulated Aβ. Few studies have focused on the improvement of tau hyperphosphorylation and neuronal loss in AD, which should thus be a focus of future research.

Vascular Dementia
Vascular dementia (VD) is the second most common form of dementia and is caused by vascular pathologies causing brain injury (Poh et al., 2021). In a rat model of VD induced by cerebral ischemia-reperfusion injury, increased angiogenesis was observed with berberine chloride (50 mg/kg) treatment, which may be due to the activation of hypoxia-inducible factor 1α (HIF-1α)/VEGF signal pathway . In general, few studies have focused on the relationship between berberine and vascular in VD. According to the development of new biological in recent years, imaging technology is conducive to strengthen the study of berberine and cerebrovascular diseases. Second near-infrared II (NIR-II) imaging, a kind of biomedical imaging technology with characteristics of high sensitivity, high resolution, and real-time imaging, can visualize the vasculature in the brain . Therefore, we can directly utilize the NIR-II to observe the improvement of the vascular in the brain by treatment with berberine.
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 more common in the elderly, and mostly manifests as simple systolic hypertension; however, it is increasing in younger age groups (James et al., 2014;Zhang and Moran, 2017). Among the many mechanisms underlying arterial stiffness, endothelial dysfunction is believed to be a critical determinant for its onset and progression (Kostov and Halacheva, 2018;Safar, 2018). Owing to arterial stiffness, elevated arterial blood pressure can contribute to both extracellular matrix (ECM) deposition and remodeling or enhanced contractility or stiffness of VSMCs (Bertorello et al., 2015). In vitro, berberine (at concentrations of 1.25, 2.5, and 5 μM) considerably inhibited aortic endothelial cells in spontaneous hypertensive rats (SHRs) by suppressing cell proliferation, apoptosis, and down-regulating the expression of Toll-like receptor 4 (TLR-4), myeloid differentiation protein 88 (MYD-88), NF-κB, IL-6, and TNF-α (Wang and Ding, 2015). Numerous studies have verified that vascular endothelial cell dysfunction and lncRNA activity may together be associated with hypertension (Lorenzen and Thum, 2016;Konukoglu and Uzun, 2017). The levels of five lncRNAs were found to be modulated by the administration of 100 mg/kg berberine in mice with hypertention, which may preserve vascular endothelial cell function . Endothelial microparticles (EMPs) are extracellular vesicles that are shed by the endothelium as a result of activation, injury, or apoptosis of endothelial cells, and are considered important biomarkers of the status of endothelial cells and vascular function (Sun et al., 2016). Endothelial progenitor cells (EPCs) mobilized from bone marrow can migrate to the peripheral blood and differentiate into mature endothelial cells, contributing to endothelial recovery (Pyšná et al., 2019). One study found that EPC number and activity was significantly reduced among people with hypertension when compared to healthy individuals, and low circulating levels of EPCs may contribute to endothelial dysfunction (Waclawovsky et al., 2021). Abnormal circulating EMPs and EPCs in SHRs were ameliorated by berberine treatment associated with endothelial dysfunction and arterial stiffness in SHRs . Moreover, the production of NO caused by oxidative stress, aging, and spontaneous hypertension increases endotheliumdependent contractions (EDCs), contributing to blunted endothelium-dependent vasodilation (Vanhoutte et al., 2005;Vanhoutte et al., 2017). Continuous ER stress can exert detrimental effects through a maladaptive, unfolded protein response, resulting in cellular defects and disturbed vascular function (Ren et al., 2021). However, incubation with 1 μM berberine has been shown to reduce EDCs by activating the AMPK pathway, thus inhibiting ER stress and ROS generation, leading to cyclo-oxygenase 2 (COX-2) downregulation in SHR carotid arteries (Liu et al., 2015). Ca 2+ signals regulate vascular function in endothelial cells; Ca 2+ release from the ER and/or Ca 2+ influx through ion channels at the endothelial cell membrane results in endotheliumdependent vasodilation and diminished vascular resistance (Cook et al., 2012;Ottolini et al., 2019;Wang et al., 2021). Notably, endothelial transient receptor potential vanilloid 4 (TRPV-4) channels are associated with hypertension, as they regulate Ca 2+ concentrations (Ottolini et al., 2020). Long-term administration of berberine has been shown to directly induce vasorelaxation, decreasing blood pressure and vascular stiffness, by suppressing the activity of TRPV-4 channels . Additionally, mechanical stretching forces increase the proliferation and apoptosis of VSMCs by activating the protein disulfide isomerase (PDI) redox system. Berberine has been shown to inhibit the PDI ER system and the MAPK pathway, thereby attenuating the simultaneous increases in VSMC proliferation and apoptosis observed in response to mechanical stretching during hypertension . Collectively, these results demonstrate that berberine may effectively ameliorate endothelial dysfunction, arterial stiffness, and vascular remodeling, resulting in reduced hypertension, and which the mechanism may be related to the inhibition of ER stress and activation of the AMPK pathway. An increasing number of studies on hypertension have focused on the Ca 2+ and TRPV-4 pathways in recent years ( Table 4).

Pulmonary Arterial Hypertension
Pulmonary arterial hypertension (PAH) is closely associated with extensive vascular remodeling, especially pulmonary arterial medial hypertrophy and muscularization, due to aberrant proliferation of pulmonary artery smooth muscle cells (PASMCs) resulting from hypoxia (Bisserier et al., 2021;Sharifi Kia et al., 2021). Hemodynamic and pulmonary pathological data showed that chronic hypoxia notably elevated the median width of pulmonary arterioles (Choudhary et al., 2011;Khoramzadeh et al., 2019). However, berberine substantially decreased pulmonary vascular remodeling in mice with hypoxia-induced PAH by inhibiting Akt/ mammalian target of rapamycin (mTOR)/HIF-1α and transforming growth factor β (TGF-β) pathways, and activating bone morphogenetic protein type 2 (BMPR-2) receptor Liu et al., 2019). Wande et al. (2020) demonstrated substantial proliferative activities in hypoxia-induced human PASMCs, were possibly mediated by berberine (at concentrations of 10 μM) via inhibiting the thioredoxin (TRX-1) and β-catenin pathways. Activation of protein phosphatase 2A (PP2A) has been shown to induce apoptosis of PASMCs in people with PAH (Ferron et al., 2011). Berberine may prominently attenuate proliferation and migration of PASMCs induced by norepinephrine, alleviating PAH via PP2A pathway . These results together show that berberine significantly suppresses pulmonary vascular remodeling and proliferation of vascular endothelial cells in PAH; the antiproliferative and antiapoptotic effects may be mediated by inhibition of the Akt/mTOR/HIF-1α and PP2A pathways ( Table 4).
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 dependent vasodilatation through the activation of the AMPK pathway (Wang et al., 2009). Advanced glycation end products (AGEs) are produced via the nonenzymatic glycation reaction and are considered major pathogenic factors that trigger vascular complications in diabetes (Azegami et al., 2021). Incubation with 5 or 10 μg/ml berberine significantly inhibited AGE formation in high-glucose-AGEs-induced micro-endothelial injuries (Hao et al., 2011). Diabetic peripheral neuropathy, characterized by perivascular neuropathy, is reported to be involved in vascular disorders associated with diabetes (Olesen et al., 2021). The mesenteric artery, a part of the splanchnic circulation system, is involved in the regulation of arterial pressure (van Dijk et al., 2020). The iliac artery is mainly responsible for blood supply to the lower extremities and pelvic organs (Maier et al., 2020). Both of these sites are predisposed to large vascular lesions in diabetes. Oral administration of 200 mg/kg berberine markedly enhanced the nitrergic neural activity in the superior mesenteric artery and diminished the adrenergic function in the iliac artery, resulting in vasodilatation in diabetic rats (Geng et al., 2016;Zhao et al., 2019b). Intracellular Ca 2+ , which is tightly controlled by Ca 2+ channels and transporters, is an important messenger in VSMC dedifferentiation (Nagel et al., 2006). Elevated concentrations of intracellular Ca 2+ is a primary stimulus for smooth muscle contraction; it is reported that diabetic vascular dysfunction is tightly coupled to the impairment of intracellular Ca 2+ processing in VSMCs (Searls et al., 2010;Yang et al., 2020). Impaired cerebral arterial vasodilation can be alleviated by berberine in a diabetic rat model via down-regulation of the intracellular Ca 2+ processing of VSMCs (Ma et al., 2016). Hyperglycemia and hypertension are the two primary risk factors for vascular disease in diabetic patients (Cryer et al., 2016;Petrie et al., 2018). Increases in intracellular Ca 2+ along with decreases in K + can lower the membrane potential and enhance the vasoconstriction response and the proliferation of VSMCs, leading to hypertension (Seki et al., 2017;Sun et al., 2021). Chronic administration of 100 mg/kg berberine reduced blood pressure and improved vasodilation in diabetic rats by activating the Ca 2+activated K + channel . Aberrant miR-133a expression in endothelial cells induces endothelial dysfunction and impaired endothelium-dependent vasodilation, aggravating the pathogenesis of cerebrovascular diseases (Li et al., 2016b). Administration of berberine (1.0 g/kg for 8 weeks) reduced miR-133a expression and impairments in learning and memory, increasing the vasodilation in the middle cerebral artery to improve VD associated with diabetes (Yin et al., 2019). Furthermore, existing evidence suggests that hyperglycemia and hypoxia can trigger cerebrovascular dysfunction (Lefferts et al., 2016;Jin et al., 2019). Berberine at a concentration of 30 μM counteracted the attenuating effects of hypoxic/high-glucose conditions on the proliferation and migration of rat brain microvascular endothelial cells, which was in part mediated by the SIRT-1/HIF-1α/VEGF pathway (Mi et al., 2019). According to these studies, berberine had a hypoglycemic effect through improving insulin resistance, and has robust anti-inflammatory, antiapoptotic and antiendothelial injury effects in DM. Berberine appears to improve endothelial dysfunction by regulating Ca 2+ and K + channels in DM (Table 5).

Diabetic Retinopathy
Diabetic retinopathy (DR) is a retinal microvascular disease caused by chronic hyperglycemia leading to angiogenesis in retina (Ai et al., 2019). In recent years, improvement of disordered glucolipid metabolism is considered one of the most effective strategies for the treatment of DM and its complications (Zhao et al., 2021b). Berberine has been shown to block DR development by modulating the glucolipid metabolism and inhibiting the HIF-1α/VEGF/NF-κB pathway (Yin et al., 2021). The pathological processes involved in DR may be related to increased levels of pro-inflammatory factors, leading to oxidative stress and the apoptotic cascade (Ai et al., 2020;Robles-Rivera et al., 2020;Gong et al., 2021). Berberine may deactivate the NF-κB pathway, thus suppressing oxidative stress and cell apoptosis in DR (Zhai et al., 2020). As a major energy receptor and metabolic regulator, AMPK is one of the therapeutic targets for metabolic and vascular diseases (Lu et al., 2019). 10 or 20 μM berberine was shown to have therapeutic effects, protecting Müller cells from 30 mM glucose-induced apoptosis by enhancing autophagy and activating the AMPK/mTOR pathway in DR . These results together indicate that berberine could attenuate the pathogenesis of DR, mainly through regulating the HIF-1α/VEGF/NF-κB and AMPK/mTOR pathways to inhibit microvascular proliferation, oxidative stress and apoptosis ( Table 5).

Diabetic Nephropathy
Diabetic nephropathy (DN), one of the most serious microvascular complications of DM, is the leading cause of end-stage renal failure (Lai et al., 2018). The early stage of DN is mainly characterized by abnormal renal hemodynamics, which mainly manifests as decreased vascular resistance in the glomerulus (Toledo et al., 2015). Research has shown that accumulation of ECM production in the glomerular mesangial membrane may be related to NF-κB pathway (Gong et al., 2020). The ameliorative effects of berberine (300 mg/kg) on ECM accumulation may be due to decreased TGF-β1 and ICAM-1 resulting from inhibition of the NF-κB pathway, as shown in a rat model of DN (Liu et al., 2010a). Recent studies have shown that abnormal levels of β-arrestins, including β-arrestins 1 and 2, have a role in microvascular permeability by regulating the production and function of ICAM-1 and vascular cell adhesion molecule 1 (VCAM-1) in the kidneys of a rat model of DN (Noh et al., 2017;Zhang et al., 2018). Orally administration of 100 or 200 mg/kg berberine had renoprotective effects owing to decreased ICAM-1 and VCAM-1 levels and increased β-arrestin 1 and 2 in kidneys of a rat model of DN (Tang et al., 2016). Additionally, inflammation in the kidney is another aggravating factor renal vascular damage in DN (Moreno et al., 2018). Berberine may attenuate LPSinduced inflammation and extracellular matrix accumulation via the NF-κB signaling pathway . Given its robust antihyperglycemic and anti-inflammatory activities, and its inhibitory effect on angiogenesis, berberine should be considered a candidate drug for DN (Table 5).
(ApoM) which is the main carrier of plasma sphingosine-1-phosphate (S1P) (Tavernier et al., 2020). Though only a small proportion of HDL contains ApoM, ApoM-bound S1P is important in maintaining vascular integrity and inhibiting vascular inflammation (Galvani et al., 2015). In a model of polymicrobial sepsis, berberine removed damaged GVB resulting from TLR4-mediated hyperglycemia, insulin resistance and proinflammatory molecule production, thus enriching ApoM gene expression and plasma ApoM via activating the ApoM/ S1P pathway (Li et al., 2020b). A previous study showed that the neonatal small intestine is prone to necrotizing enterocolitis (NEC), a severe acquired disease characterized by inflammation (MohanKumar et al., 2019). However, these changes were notably reversed by treatment with berberine; the anti-inflammatory mechanism of berberine in this context may act via suppression of the PI3K/Akt pathway . Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical morbidity (Tsai et al., 2018). In the normal healing process, ECM can be completely degraded by the proenzyme MMP (Altoé et al., 2021). Berberine prevented adhesion reformation, promoting the activation of MMP-3 and MMP-8 by directly blocking tissue inhibitor of metalloproteinase-1 (TIMP-1) activation in fibroblasts . To summarize, berberine can inhibit endothelial and gut vascular permeability, inflammation and adhesion reformation in intestine; it may act through several pathways, including the WNT/β-catenin, ApoM/S1P and PI3K/Akt pathway ( Table 6).

Vasculature in Cancer
Angiogenesis is a process in which new blood vessels form and grow from pre-existing vessels; this process occurs under healthy and pathological conditions, such as cancer (Fang et al., 2021).
VEGF promotes angiogenesis in endothelial cells. Berberine has been shown to suppress angiogenic action, HUVEC proliferation and migration, by inhibiting VEGF, against cancerous Meth A cells and hepatocellular carcinoma (Jie et al., 2011;Yahuafai et al., 2018). VEGF-2 is a major mediator of the biological effects of VEGF, and therefore plays an important role in tumor angiogenesis (Sharaky et al., 2020). Berberine was shown to inhibit angiogenesis in glioblastoma xenografts by targeting the VEGFR-2/ERK pathway (Jin et al., 2018). In tumor development, chronic inflammation leads to a sharp increase in VEGF expression (Crusz and Balkwill, 2015). Elevated inflammatory cytokine levels and epithelial-mesenchymal transition in glioma cells were shown to be reversed by 100 μM berberine; the mechanism for this may be via suppressing ERK-1/2 signaling and production of IL-1β and IL-18 (Tong et al., 2019). VEGFR-3 in lymphatic endothelial cells were essential for the development of VSMCs (Antila et al., 2017). Clinically, elevated levels of VEGFR-3 are thought to be correlated with cancers resulting from metastasis via the lymph nodes, such as renal carcinoma (Guida et al., 2014). Following an inflammatory stimulus, the production of ROS by oxidative stress and energy deficit at mitochondrial, lysosomal, or ER loci can directly lead to irreversible damage of tumor cells and initiate cell apoptosis and autophagy (Reuter et al., 2010). Combined with photodynamic therapy, 20 μM berberine triggered metabolite changes in renal carcinoma cells, resulting in inhibited cell proliferation, tumorigenesis, and angiogenesis, and inducing autophagy and apoptosis via increased ROS generation (Lopes et al., 2020). Another study implied that 12-O-tetradecanoyl phorbol-13-acetate (TPA) significantly increased the level of VEGF and fibronectin in both MCF7 and T47D breast cancer cells (Kim et al., 2009). TPA-induced VEGF and fibronectin expression was decreased by berberine treatment, via inhibition of the PI3K/A pathway, in breast cancer cells (Kim et al., 2013). Taken together, these results show that berberine may effectively inhibit the proliferation and angiogenesis of tumor cells by inhibiting VEGF, ERK and PI3K/Akt pathways and promote the apoptosis of tumor cells (Table 7).

Other Vascular Diseases
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that may involve angiogenesis, particularly during the earliest stages of the disease (Yang et al., 2018;Lyu et al., 2021). Angiogenesis is strictly regulated by several pro-and antiangiogenic factors including VEGF, which have been suggested to be involved in neovascularization in RA joints (Li et al., 2018a). Berberine was shown to have anti-inflammatory and antiangiogenic effects in a rat model of RA by decreasing the level of inflammatory factors, and suppressing p-ERK, p-p38 and p-JNK activation . Additionally, it is reported that doxorubicin-induced vascular congestion and inflammatory cell infiltration in the liver were largely attenuated by berberine pretreatment (Zhao et al., 2012).
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762654 and its derivatives have been rarely investigated. Considering the long-term development for berberine prevention and treatment of vascular diseases, comprehensive toxicity investigations, especially potentially cumulative toxicity in vivo studies, need to be carried out. It is also necessary to explain the efficacy and toxicity of berberine for use in human pharmacokinetic studies, and the identification of the ideal dosage are of enormous significance if side effects associated with drug accumulation are to be avoided. Relatively newly developed biological techniques, including microfluidic technology, computational toxicological methods, hepatoid cell models, and highthroughput chip models, could be employed to explore the toxicity of berberine (Banerjee et al., 2016;Kimura et al., 2018). In addition, single-cell in vitro models, as reported in the literature reviewed here, may not adequately reflect the pathogenesis of diseases in vivo. A multi-organ in vitro model based on microfluidic technology may be more helpful models of vascular diseases for the evaluation of the safety and efficacy of berberine (Rothbauer et al., 2018;Malik et al., 2021). For the clinical trial of berberine on vascular disease, many factors limit its clinical application, including the low methodological quality and drug-drug interactions (Imenshahidi and Hosseinzadeh, 2019). Therefore, the development of a standardized dosage, administration route, duration and adverse reaction of berberine could also be pursued in clinical settings for better therapeutic efficacy and safety. Concurrently, we should strengthen the mechanism of berberine in the treatment of vascular diseases. Novel technologies that could be employed to explore the mechanism of action of berberine in the treatment of vascular diseases include CRISPR-CAS-9 gene editing, metabolomics, proteomics, and genomics. Most importantly, recently published investigations of the pharmacological mechanisms of berberine may provide new insights into the treatment of vascular diseases by berberine. For example, Zhao and others demonstrated that 560 mg/kg berberine administered orally in Coptis chinensis can significantly increase insulin secretion via the potassium voltage-gated channel subfamily H member 6 (KCNH-6) potassium channel in mice with HFD-induced hyperglycemia (Zhao et al., 2021a). Overall, this study comprehensively reviewed and summarized the pharmacokinetics properties and therapeutic potentials of berberine in diverse vascular diseases, thus providing experimental evidence for future research to discover novel drugs from Chinese medicine monomers.

AUTHOR CONTRIBUTIONS
XM, FL, and XA conceived the study; XA, PY, LL, SL, and XL collected, analyzed, and interpreted the relevant literatures; XA, LP, and JL drew all the figures and tables; XA wrote the manuscript; XM and FL supervised the study and revised the manuscript. The final version of the manuscript was read and approved by all authors.