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Circulating metabolomics profiling reveals novel pathways associated with cognitive decline in patients with hypertension

Background: Hypertension is a significant risk factor for cardiovascular disease, and it is associated with dementia, including Alzheimer’s disease (AD). Although it may be correlated with AD in terms of symptoms, the link between hypertension and AD pathological biomarkers, and the potential underlying mechanism of hypertension with cognitive decline, are still not well understood. Methods: The Mini-Mental State Examination (MMSE) scores were used to evaluate cognitive function. Enzyme-linked immunosorbent assays were used to examine plasma amyloid-beta (Aβ)40, Aβ42, and tau concentration in hypertensive patients. Metabolomics and metagenomics were performed to identify the significantly changed circulating metabolites and microbiota between healthy individuals and hypertensive patients. Pearson’s correlation was used to examine the association between cognitive indicators and differential metabolites. Results: We found significantly decreased MMSE scores, elevated plasma Aβ40, and decreased Aβ42/Aβ40 ratio in hypertensive patients, which are critically associated with AD pathology. Based on metabolomics, we found that significantly altered metabolites in the plasma of hypertensive patients were enriched in the benzoate degradation and phenylpropanoid biosynthesis pathways, and they were also correlated with changes in MMSE scores and Aβ42/Aβ40 ratio. In addition, metabolomics signaling pathway analysis suggested that microbial metabolism was altered in hypertensive patients. We also identified altered blood microbiota in hypertensive patients compared with the controls. Conclusions: Our study provides a novel metabolic and microbial mechanism, which may underlie the cognitive impairment in hypertensive patients.


Introduction
Dementia is a global health problem that affects about 47 million people worldwide. This number is expected to double by 2030 reaching 75 million, and the number of dementia cases are estimated to almost triple by 2050 reaching 135.5 million. 1 Although the understanding of the relationship between cardiovascular dysfunction and brain health has significantly improved over the last decades, whether hypertension constitutes a potentially treatable risk factor for cognitive decline and dementia remains a subject of debate. Hypertension is highly prevalent and is a risk factor for cardiovascular disease as it can induce microvascular dysfunction and atherosclerosis, which can result in cerebral hypoperfusion, microhemorrhages, and cerebral small vessel disease. 2 Recently, several clinical trials, such as the Systolic Blood Pressure Intervention Trial (SPRINT) Memory and Cognition in Decreased Hypertension (SPRINT-MIND) supported by the National Institute of Health and Hypertension Cardiovascular Outcome Prevention and Evidence Asia Network, have tried to determine the association between blood pressure (BP) control and dementia. 3,4 SPRINT-MIND study suggests that lowering BP actually has a measurable impact on mild cognitive impairment (MCI). 5 It has been reported that hypertension in patients with Alzheimer's disease (AD) is associated with worse cognitive function, increased neuropsychiatric burden, and lower hippocampal glucose metabolism. 6 In addition, pulse pressure elevation is also related to increased phosphorylated tau and decreased amyloid-beta (Aβ) 42 , the most important pathological hallmarks of AD, in the cerebrospinal fluid (CSF) of cognitively normal older adults. 7 Nevertheless, the link between hypertension and AD biomarkers is still not well understood. Metabolomics is widely used to evaluate metabolic changes associated with disease and to explore disease biomarkers; it has also been utilized to characterize risk factors for cardiovascular diseases, including hypertension. 8 However, there is still no evidence showing whether metabolic changes contribute to hypertension with cognitive decline.
In this study, we evaluated the cognitive function and circulating metabolites and microbes in patients with hypertension. We found decreased Mini-Mental State Examination (MMSE) scores and Aβ 42 /Aβ 40 ratio in hypertensive patients, which were correspondingly correlated with the significantly changed circulating metabolites. In addition, there appeared to be altered blood microbiota in hypertensive patients compared with the controls. Hence, our study provides a novel metabolic and microbial mechanism, which may underlie cognitive impairment in hypertensive patients.

Material and methods
This study complied with the principles of the Declaration of Helsinki and was approved by the ethics committees of the institutional review board at Shunde Hospital, Southern Medical University, China (approval number: 20190903). Written informed consent was obtained from the patients or their legal guardians, and all research was performed in accordance with the relevant guidelines and regulations.

Study populations
A total of 33 hypertensive patients and 36 ageand sex-matched healthy individuals (controls) were enrolled from Shunde Hospital, Southern Medical University (Guangzhou, China) (As shown in Table 1). Patients with heart failure, acute myocardial infarction, undergoing medical treatment for dementia, sepsis, history of malignancy, autoimmune disease, secondary hypertension, severe renal failure [estimated glomerular filtration rate (eGFR) < 15 ml/min/1.73 m 2 or under renal replacement therapy], severe anemia (Hb <60 g/L), or severe hepatic disease [cirrhosis or bilirubin >3× upper limit of normal (ULN) or aspartate aminotransferase/alanine aminotransferase/alkaline phosphatase >5× ULN] were excluded.

Hypertension diagnosis
Hypertension was defined as systolic BP ⩾ 140 mmHg and/or diastolic BP ⩾ 90 mmHg, according to current Chinese guidelines for the management of hypertension. 9

MMSE evaluation
The MMSE scale was used to assess cognitive function. The MMSE scores ranged from 0 to 30, and higher scores indicated better cognitive function. Patients were asked to perform the MMSE on the day of enrollment, guided by a skilled nurse who had been trained for the task.

Human plasma collection
Fasting venous blood samples were collected to measure levels of glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), and creatinine using the Olympus AU2700 Automatic Biochemical Analyzer (Olympus, Tokyo, Japan). Low-density lipoprotein cholesterol (LDL-C) was calculated using Friedewald equation. High sensitivity C-reactive protein (hs-CRP) was determined by the high-sensitivity nephelometric method. After centrifugation at 1,500 g for 10 min at 4°C, plasma samples were stored at −80°C for future measurements of Aβ 40 and Aβ 42 .

Metabolites extraction
The plasma samples were incubated on ice for 5 min and then were centrifuged at 15,000 rpm at

Untargeted metabolomics data analysis
The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.0 (CD 3.0; Thermo Fisher Scientific) to perform peak alignment, peak selection, and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, 1,00,000. Then the peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Finally, the peaks were matched with the mzCloud (https:// www.mzcloud.org/) and ChemSpider (http:// www.chemspider.com/) database to obtain accurate qualitative and relative quantitative results.

Statistics
Continuous variables are presented as the mean ± standard deviation (SD) or median and interquartile range. Categorical variables are presented as the number and percentage. Baseline characteristics of hypertensive patients and controls were compared by the two-tailed t-test for normally distributed continuous variables, Wilcoxon rank-sum test for non-normally distributed continuous variables, and the chi-square test for categorical variables. The Pearson productmoment correlation coefficient (r) was calculated as a measure of linear association between MMSE and tau, and Aβ 40 and Aβ 42 in the controls and hypertensive patients, respectively, adjusting for age, smoking history, fasting plasma glucose (FPG), TG, TC, HDL-C, LDL-C, hs-CRP, and eGFR. Statistical analysis of the data was performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). All of the p-values were two-tailed and values of p < 0.05 were considered to be statistically significant.

Baseline characteristics
To explore the metabolic change and its correlation with cognitive function, we included 36 2.92 ± 0.80 mmol/L, respectively) (p = 0.023 for eGFR and p = 0.039 for LDL-C, respectively; Table 1). Other baseline characteristics such as age, gender, HbA1c, and hs-CRP showed no obvious difference between these two groups.
Hypertension is associated with a worse cognitive function Given that high BP is a risk factor for cognitive decline, we examined plasma Aβ 40 , Aβ 42 Figure 1A). In addition, we performed MMSE evaluation and found that MMSE scores were decreased in hypertensive patients compared with healthy controls (P = 0.0247; Figure 1D), which was consistent with our ELISA results showing a decreased Aβ 42 / Aβ 40 ratio in hypertensive patients. Next, we performed Pearson's correlation analysis to further evaluate the correlation among plasma Aβ 40 , Aβ 42 , tau concentrations, and MMSE evaluation. We found that plasma Aβ 40 and tau concentration were negatively correlated with MMSE scores, while the Aβ 42 /Aβ 40 ratio was positively correlated  Figure 1B), suggesting that these parameters may be indicators for cognitive evaluation. Importantly, in hypertensive patients, we found a close correlation among plasma Aβ 40 , Aβ 42 /Aβ 40 ratio, and MMSE scores ( Figure 1F, J), which was consistent with altered Aβ 40 concentration and Aβ 42 /Aβ 40 ratio in patients with hypertension. However, we did not find a correlation between plasma Aβ 42 concentration and MMSE scores in healthy controls ( Figure 1G), or a correlation between plasma Aβ 42 , tau concentration and MMSE scores in hypertensive patients ( Figure  1H; Supplemental Figure 1C). These results suggest that the increased Aβ 40 concentration and decreased Aβ 42 /Aβ 40 ratio were closely correlated with MMSE evaluation, and cognitive impairment existed in hypertensive patients in our study.

Metabolic landscape in hypertensive patients and controls
Because dysfunctional metabolism contributes to hypertension, in this study we performed LC-MS/ MS to reveal the metabolic changes in hypertensive patients. A distinct separation of metabolites between controls and hypertensive patients was found by both three-dimensional principal components analysis and partial least squares-discriminant analysis (PLS-DA) score plots in the positive and negative ion mode, which indicated the significantly different metabolic profiles (Figure 2A-F). A total of 2604 compounds were identified in the hypertensive patients and healthy control individuals through metabolomics. Based on the variable importance in projection (VIP > 1.0) calculation through PLS-DA, we listed the significantly changed metabolites. In the positive ion mode, 28 compounds were increased and 231 compounds were decreased ( Figure 2G); while in the negative the ion mode, 21 compounds were increased and 66 compounds were decreased ( Figure 2H). Thus, a total of 49 and 297 compounds including metabolites, pollutants, medicines, and food additives had significantly increased or decreased abundance. To further evaluate the metabolic similarities and differences of each significantly biosynthesized metabolite, correlation analysis and hierarchical cluster analysis approaches were conducted. Correlation analysis of differential metabolites is shown in Figure 2I and J. Figure 3A shows that the metabolism markedly differed between the patients and the control group. Then the changed metabolites were subjected to the known metabolic relation network database, namely, Kyoto Encyclopedia of Genes and Genomes (KEGG). KEGG results suggested that the downregulated metabolic network primarily consisted of the benzoate degradation pathway, phenylalanine metabolism, tyrosine metabolism, phenylpropanoid biosynthesis, citrate cycle, and phospholipid metabolism ( Figure 3B, C).
We showed the global metabolic network in which the changed metabolites in hypertensive patients compared with healthy individuals were involved (Figure 4). At the pathway and network levels, benzoate and acetyl-CoA were the key metabolites connected to different pathways. The identified compounds with a benzene ring were converted to benzoate before degradation through the citrate cycle. We showed the representative changed compounds enriched in the benzoate degradation pathway between controls and hypertensive individuals ( Figure 5A-O). These compounds, containing phenylglyoxylic acid, benzoate, naphthalene, 1-naphthol, phenanthrene, phenol, 2,3-naphthalenediol, biphenyl, guaiacol, eugenol benzoate, cis,cis-muconic acid, 4-hydroxy-3methylbenzoic acid, hydroquinone, 3-butylidenephthalide and naproxen, were significantly decreased in the plasma of hypertensive patients ( Figure 5A-O). For phenylpropanoid biosynthesis, we listed the important metabolites involved in phenylpropanoid biosynthesis ( Figure 6A-I). We found that caffeate (caffeic acid), caffeic acid phenethyl ester, vanillin, 4-methoxycinnamaldehyde, scopoletin, eugenol, 3,4-dimethoxycinnamic acid, ethyl methoxycinnamate, and cinnamaldehyde were significantly decreased in hypertensive patients compared with the controls (Figure 6A-I).

Correlation analysis between the altered metabolites and cognitive impairment in hypertensive patients
Because we found significantly changed metabolites in hypertensive patients, and we also found a cognitive decline in these hypertensive patients, we examined the correlation of representatively changed metabolites with cognitive indicators in hypertensive patients using Pearson's correlation. We found that the representative metabolites, benzoate, naphthalene, phenanthrene, phenol, 2,3-naphthalenediol, guaiacol, eugenol benzoate, 3-butylidenephthalide, and naproxen, which were  were statistically significantly positive correlated with MMSE (Supplemental Figure 3A-F, H, I), with the exception of 3,4-dimethoxycinnamic acid (Supplemental Figure 3G). Since Aβ 42 /Aβ 40 ratio is a promising evaluation of cognitive function, we also correlate these representative metabolites with Aβ 42 /Aβ 40 ratio. Here, our results showed that Phenylglyoxylic acid, Naphthalene, 1-Naphthol, Phenanthrene, 2,3-Naphthalenediol, Biphenyl, Guaiacol, Eugenol benzoate, and Naproxen, which were enriched in the benzoate degradation pathway, were statistically significant positive related with Aβ 42 /Aβ 40 ratio ( Figure 7A Figure 8A, C). Thus, our results confirmed that the abundance of key metabolites enriched in the benzoate degradation and phenylpropanoid biosynthesis pathways were significantly associated with MMSE score and Aβ 42 / Aβ 40 ratio, which suggest that these key metabolites may contribute to the cognitive decline we observed in hypertensive patients.

Circulating microbiota in hypertensive patients
Because our metabolomic KEGG results revealed that the changed metabolites were enriched in microbial metabolism in diverse environments ( Figure 3B, C) and circulating microbiota contributes to the pathogenesis of hypertension, 12 we further examined the microbial communities at the whole blood level using metagenomic approaches. We found that nearly 100 species of microbes were identified in the whole blood samples. At the phylum level, Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria, and Actinobacteria were the highly abundant microbes, occupying about 90% of the total abundance in samples of both patients and the controls (Supplemental Figure 4). We also found that β-diversity was decreased in the hypertensive group compared with the controls (p = 0.018; Figure 9A), and the principal coordinates analysis plots of these two groups are shown in Figure 9B. However, αdiversity, including the observed species, Chao1, ACE, Shannon, Simpson, and Phylogenetic diversity (PD) whole tree showed no obvious changes between controls and hypertensive patients  Figure 5A-F). Next, we used 16S rDNA gene sequencing to determine the alterations in blood microbiota composition between healthy and hypertensive patients. The taxon abundance of each sample was classified in terms of phylum, class, order, family, genus, and species levels, mainly using the small subunit ribosomal RNA reference database. The hierarchy cluster heat-map revealed the top 35 most abundant differentiated taxa ( Figure 9C), and the abundance of top 10 species at the genus level in the blood is shown in Figure 9D. We also performed a t-test comparison to show the conspicuous group differences at the genus levels. According to KEGG pathway annotation, the number of different genes enriched in the different pathways is shown (Supplemental Figure 6A). We also revealed the relative abundance of top 10 functional pathways at the genus level between controls individuals and hypertensive patients (Supplemental Figure  6B). The functional prediction based on FAPROTAX analysis confirmed that the microbes accessed the patient circulating system primarily through gastroenteritis, diarrhea, pneumonia, and intracellular parasites (Supplemental Figure 6C). The reported datasets related to gut microbiota confirmed that Bacteroidetes and Firmicutes dominated the adult gut microbiota followed by Actinobacteria, Verrucomicrobia, and Proteobacteria. 13 Those results were consistent with the abundance of microbes in the current study, which confirmed that the blood microbiota journals.sagepub.com/home/tan 11 primarily stemmed from the gut. We found representative changes in six blood microbiota at the genus levels, and found that Stenotrophomonas and Turicibacter were significantly decreased in the whole blood of hypertensive patients compared with the controls (Figure 9E, F), whereas Streptococcus, Lactococcus, Parabacteroides, and Helicobacter were significantly increased in hypertensive patients compared with the controls (Figure 9G-J).

Hypertension is associated with cognitive decline and pathological characteristics in AD
Recently, some groups reported that dementia was highly associated with cardiovascular disease, such as coronary heart disease and congestive heart failure. 14,15 Hypertension is a significant risk factor for cardiovascular disease, and they share aging as the most important risk factors. Thus, hypertension is closely correlated with cognitive decline, and vice versa, cognitive impairment is related to hypertension severity. 16,17 Moreover, antihypertensive approaches show potential benefits in improving cognitive function in elderly hypertensive patients. 18 Importantly, several studies have explored the underlying mechanisms between high blood pressure and cognitive impairment, and the evidence may lie on the disruption of the neurovascular unit, age-related insulin-like growth factor 1 deficiency, neurovascular oxidative stress, and neuroinflammation. [19][20][21] Intriguingly, during aging, high blood pressure reduced cerebral hemodynamics and connectivity and led to cognitive impairments in a genetic AD mouse model. 22 In addition, Nazarian et al. also reported the genetic association between AD and hypertension. 23 However, the metabolic mechanisms in the relationship between hypertension and cognitive impairment have yet to be fully elucidated.
In this study, we found that plasma Aβ 40 was increased and Aβ 42 /Aβ 40 ratio was decreased in subjects with hypertension. It is well established that extracellular deposition of amyloid plaques and intracellular neurofibrillary tangles (NFTs) are the two major hallmarks of AD. Aβ 40 and Aβ 42 , the main components of the amyloid plaque, are derived from the amyloid precursor protein, which is cleaved by β-secretase and γ-secretase. In physiological conditions, Aβ 40 is the most abundant species, while Aβ 42 is the most toxic due to its high hydrophobicity; Aβ 42 has a high tendency for aggregation. Usually, Aβ is cleared through the blood-brain barrier (BBB), and plasma Aβ 40 level is much higher than Aβ 42 .
In this study, we found elevated plasma Aβ 40 rather than Aβ 42 in subjects with hypertension compared with the controls, consistent with a previous report. 24 Although Aβ deposition in the brain is the pathological hallmark of AD, a lower plasma Aβ 42 /Aβ 40 ratio was recently found to be highly associated with AD dementia. [25][26][27][28] Circulating Aβ 42 /Aβ 40 ratio (plasma or CSF) could reflect the function of the BBB, which is responsible for Aβ clearance. However, as a more easily detectable method, circulating Aβ 42 /Aβ 40 ratio could predict the MCI and prevent the aggravation of AD dementia. In this study, we also found that the plasma Aβ 42 /Aβ 40 ratio was decreased in patients with hypertension, which was closely correlated with decreased MMSE scores in these patients. Thus, our results indicate that cognitive decline may occur in hypertensive patients, consistent with other reports that hypertension induces cognitive impairment and memory deterioration. [29][30][31]  Regarding the potential mechanism, we conclude that, on one hand, high BP causes several acute effects, and the most established in the brain is stroke. In this case, high BP disrupts the neurovascular units via breakdown of cerebral vasculature, leading to brain damage and cerebral degeneration. 2 In addition, hypertension damages the BBB, impairs microvascular deposition and clearance of Aβ and NFTs through the BBB, and induces the production of Aβ and NFTs. 32,33 Moreover, dietary factors also contribute to cognitive decline and vascular disease, which may involve hypertension via gut-brain communication. 34 Benzoate degradation and phenylpropanoid biosynthesis are the most altered pathways in hypertensive patients In this study, we found that the key metabolites enriched in benzoate degradation and phenylpropanoid biosynthesis were decreased in hypertensive patients. Pollutants with an aromatic ring were converted in the pathway of benzoate degradation connected citrate cycle through salicylate and acetyl-CoA. Among these aromatic compounds, polycyclic aromatic hydrocarbons phenanthrene, anthracene, and naphthalene are regarded as the priority control pollutants by both the U.S. Environmental Protection Agency and the European Environment Agency because of their toxicity and persistence. The primary sources of these compounds' exposure to humans are polluted air, from ambient air and indoor air, and foodstuffs, especially those prepared through cooking methods of baking, frying, and grilling. 35 Because of the similar quality of ambient air for people living in the same district, the low abundance of polycyclic aromatic hydrocarbons and their metabolites 1-naphthol and 2,3-naphthalenediol in the samples of hypertensive patients was primarily due to the low intake of grilled foods, followed by low inhalation exposure to tobacco smoking. Other aromatic compounds, including 4-hydroxy-3-methylbenzoic acid, 4-methoxybenzoate, and eugenol benzoate, were converted to benzoate by the catalysis of oxygenases or through anaerobic respiratory chains using Fe(III) or sulphate as terminal electron acceptors. 36 For compounds in the pathway of phenylpropanoid biosynthesis, caffeate is a major phenolic phytochemical in coffee with anti-inflammatory properties, the ability to suppress DNA methylation in cancer cells, and inactivation of several ways participating in tumorigenic processes. Caffeate significantly inhibits intracellular superoxide anion generation and cell proliferation induced by angiotensin II, which stimulates proliferative and hypertrophic growth in vascular smooth muscle cells. 37 Furthermore, caffeate inhibits tyrosine phosphorylation. The decreased abundance of this compound leads to the high risk of damage in the DNA and target proteins of hypertensive patients. This inference was supported by the direct evidence that the abundance of its phenethyl derivatives, such as phenethyl caffeate and caffeic acid phenethyl ester, with similar function also decreased. Caffeic acid phenethyl ester, an active polyphenolic component of honeybee propolis, has been demonstrated to have many medicinal properties, such as antitumor and neuroprotection. Phenethyl caffeate has a role as an antineoplastic agent, antiinflammatory agent, immunomodulator, antioxidant, neuroprotective agent, antiviral agent, and antibacterial agent. Coffee is a mixture of compounds containing flavonoids, chlorogenic acid, melanoidins, and various lipid-soluble compounds. It is difficult to make comparisons between studies of coffee consumption from different regions due to different preparation methods and components of coffees. Some studies have suggested that coffee consumption increases BP, whereas the SUN Project confirmed that consumption of regular coffee can reduce the risk of hypertension among women. 38 The aromatic amino acid biosynthesis pathway is one of the core metabolic pathways that lead to the production of aromatic compounds, including phenylalanine and tyrosine. 39 Phenylalanine is the key metabolite connected with tyrosine metabolism, the benzoate degradation pathway, and ethyl methoxycinnamate transformation. The abundance of phenylalanine is stable and that of its intermediates phenylethyl alcohol and phenylglyoxylic acid was decreased in the current study, suggesting that the generated phenylalanine was prioritized for protein biosynthesis. In this study, based on circulating metabolomics, we identified the differential metabolites enriched in the pathway of tyrosine metabolism, and also found that some medicinal compounds and food additives were changed in hypertensive patients; these are listed in the Extended Data.

Cognitive decline in hypertensive patients may be associated with metabolic changes
We also found that some metabolites enriched in the benzoate degradation and phenylpropanoid biosynthesis pathways were correlated with MMSE and Aβ 42 /Aβ 40 ratio. Several compounds reportedly regulate cognitive function; for example, benzoate has been shown to improve cognitive journals.sagepub.com/home/tan 15 function in patients with chronic schizophrenia. 40 Because the phenylpropanoid pathway is suggested to be involved in modulating cognitive function, 41 caffeate and caffeic acid phenethyl ester have been proven to protect cognitive deficit in the animal model of AD and cerebral ischemia by regulating neuroinflammation and inhibiting lipid peroxidation and nitric oxide production. [42][43][44][45] In addition, vanillin has been shown to attenuate scopolamine-induced cognitive impairment via modulating the DNA-binding protein inhibitor ID-1 and neuronal differentiation. 46,47 Ethyl methoxycinnamate reportedly improves cognitive impairment independent of direct acetylcholinesterase and glutamate receptor inhibition in an aluminum-induced memory deficit rat model. 41 Moreover, cinnamaldehyde attenuates cognitive deficits via regulating extracellular signal-regulated kinase signaling in the prefrontal cortex. 48 Taken together, most of the metabolites enriched in the phenylpropanoid pathway were involved in improving cognitive performance, and according to its significant correlation with MMSE and Aβ 42 /Aβ 40 ratio, we conclude that this pathway may contribute to the cognitive decline in hypertensive patients. Though little is known about the role of metabolites in the benzoate degradation pathway in regulating cognitive function, considering their distinct correlation with MMSE and Aβ 42 /Aβ 40 ratio, further studies are needed to clarify these compounds in regulating cognitive performance.

Circulating microbiota reveals a novel underlying mechanism in hypertension
The relative abundance of microbes with the function of aromatic compound degradation was significantly higher in the hypertension group. This result is partially responsible for the lower abundance of aromatic compounds in patient blood. For sulfur transformation, the function of microbes, including Geobacter, Thiobacillus, Desulfovibrio, Ferruginibacter, and Acidibacter, in patients and healthy individuals is related to the respiration of sulfur compounds and respiration of sulfate. Therefore, the metabolism of organic compounds containing sulfur is enhanced in hypertensive patients, which is partially responsible for the decrease in abundance of methylsulfonylmethane, methionylserine, 3-thiotolene, 3-mercaptopropane-1,2-diol, S-3-oxodecanoyl cysteamine, laurilsulfate, 2-ethylhexyl hydrogen sulfate, 3,4-dehydrothiomorpholine-3-carboxylic acid, and S-3-oxodecanoyl cysteamine. In the control samples, microbes for sulfur respiration primarily used sulfate as an electron acceptor to oxidize organic carbon compounds or hydrogen under anaerobic conditions with the production of aqueous sulfide. 49 Bioconversions of nitrogen by nitrogen-transforming microorganisms are often depicted as a cycle consisting of distinct processes that proceed in an orderly fashion. 50 In the current study, microbes in the blood of patients for nitrogen metabolism were primarily involved in nitrite ammonification and nitrogen fixation. In the nitrite ammonification processes, nitrite was converted to ammonia through the reaction equations 1-4.
Dinitrogen in blood is further reduced into ammonia via N 2 fixation (equation 4). Based on these reactions, the abundance of nitrite in the patient's blood would decrease. In the control samples, the functions of microbes were primarily associated with nitrate reduction, nitrogen respiration, and nitrate respiration. Nitrate reduction is the reaction catalyzed by nitrate reductase with the conversion of NO 3 − to NO 2 − . 51 Nitrate respiration processes proceed by using nitrate as an electron acceptor at low oxygen concentrations resulting in the production of dinitrogen. The absence of the microbes with ammonia oxidation and nitrite oxidation functions confirms that these microbes dwelled in anaerobic conditions, for example, in the gut. Some microbes that trigger gastroenteritis and diarrhea are the direct evidence that verified the above inference. An analysis clarified that a nitrate-rich diet is effective in normalizing BP values. 52 Modulating the nitrate/nitrite-reducing bacterial communities to increase nitrite production is a potential solution to treat hypertension. Therefore, a lower nitrite/ nitrate ratio in the patient's blood in the current study is harmful to the patient.

Conclusion
In this study, we showed cognitive impairment in hypertensive patients, and based on the metabolomics and microbiome, we identified the differential circulating metabolites and microbiota between controls and hypertensive patients. Collectively, our results provide a novel metabolic and microbial mechanism that may underlie cognitive impairment in hypertension, and the altered metabolites and microbiota may be potential therapeutic targets in treating hypertension.