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Protective effects of 4, 5-O-dicaffeoylquinic acid against mouse sepsis via down-regulation of TNF-α, IL-6 and IL-8

Purpose: To investigate the protective effects of 4, 5-O-dicaffeoylquinic acid (DCQA) isolated from Xanthium sibiricum Patr. against mouse sepsis caused by cecal ligation/puncture (CLP) in vivo, as well as the molecular mechanisms of action involved. Methods: DCQA (7.5, 15, and 30 mg/kg/day) were administered to the mice with sepsis and the survival rate was obtained. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 were examined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the lipopolysaccharide (LPS) neutralizing ability of DCQA (2, 4, and 8 μg/mL) was measured using limulus amebocyte lysate (LAL) test in vitro. Furthermore, the effect of DCQA (10, 20, and 40 μg/mL) on mRNA expression of TNF-α, IL-6, and IL-8 in LPS (100 ng/mL)-treated RAW 264.7 cells was assessed using quantitative real time-polymerase chain reaction (RT-qPCR) assay. Results: DCQA significantly improved the survival rate of mice with sepsis caused by CLP (35, 50, and 65 %, respectively vs. 15 % for control, p < 0.05). LPS levels fell on co-incubation with DCQA in vitro. Moreover, ELISA and RT-qPCR results revealed that DCQA treatment lowered tendency in the mRNA expression of TNF-α, IL-6, and IL-8 (p < 0.01). Conclusion: DCQA exhibits protective effects against sepsis in mice mediated by downregulating TNFα, IL-6, and IL-8. Further studies, in animals and humans are requied to determine the safety and efficacy of DCQA in both animal and clinical management of sepsis.


INTRODUCTION
Sepsis is the common cause of death and a rapid-developing critical condition with a high mortality rate (> 20 %) in the intensive care units (ICU) of hospitals [1,2]. It is well-known that sepsis, generally caused by dysregulated inflammatory response to micro-organism infection, is characterized by life-threatening organ dysfunction and circulatory disturbance [3,4]. However, the related pathogenesis of sepsis is not completely clear yet, and the effective treatments are currently lacking. Therefore, it is necessary for us to develop an effective therapy to curing/attenuating sepsis.
Increasing evidence indicated that plant-derived extracts/compounds can be used as the potential resources for treatment of sepsis with few sideeffects [5,6]. Xanthium sibiricum Patr., a specie of the family Asteraceae, is widely distributed in the Chinese provinces of Anhui, Shandong, Jiangxi, and Hubei [7,8]. It was reported that about 170 phytochemical compounds have been isolated and identified from this plant, including essential oil (sesquiterpene lactones and alkene), glycoside, phenol, alkaloid, and fatty acid [9,10]. The fruits of X. sibiricum, also called Cangerzi in Chinese, are commonly used for treating inflammatory diseases, such as acute and chronic rhinitis, itching diseases and urticaria [11,12].
As continuing efforts on separating constituents of X. sibiricum, 4, 5-O-dicaffeoylquinic acid (DCQA, Figure 1), a major phenylpropanoid derivative isolated from X. sibiricum, have been found to possess an anti-inflammatory property. In this study, the protective effects of DCQA against CLP surgery-induced sepsis in mice, the effect of DCQA on the viability of LPS-treated RAW 264.7 cells, as well as the underlying molecular mechanisms were examined.

Animals
The animal experiments were carefully performed according to the Institutes of Health Guide for the Care of Animals [13], and were approved by the Committee for Animal Use of Wenzhou Central Hospital (approval no. 20170512ky-04). Male ICR mice (20 ± 2 g, 5-6 weeks) obtained from Shanghai laboratory animal center (Shanghai, China) mice, were kept in plastic pages (25 ± 2 °C; 55 ± 5 % humidity). They were adapted for 7 days with free diet and distilled water.

Preparation of CLP model of mice
The CLP mice were established as previously described [3]. Briefly, after the mice were anesthetized with isoflurane (3 mL/kg), cecum was exposed by laparotomy under sterile condition. Then, the cecum was exteriorized and ligated with a silk suture, after which it was punctured with an 18-guage needle. Following with reposition of cecum, the abdominal incision was closed with double sutures.

TNF-α and IL-6 levels in sepsis mice
The mice were sacrificed after CLP operation for 96 h, and 2 mL of blood sample was collected from each mouse. After centrifuging (10 min, 4 °C), the plasma was used for evaluation of TNFα and IL-6 using ELISA kits.

Neutralizing effect of DCQA to LPS
To measure the neutralizing ability of DCQA to LPS in vitro, the LAL test was performed. Shortly, the DCQA at three concentrations of 2, 4, and 8 μg/mL were incubated with LPS of 1 ng/mL at 37 °C, and then the LAL reagent was supplemented to the mixture with equal amount. After mixing, the kinetic turbidity of the mixture was evaluated using the Tube Reader (Jinshan Biotech, Beijing, China).

Cell viability assay
RAW264.7 cells were seeded at 8 × 10 3 cells/well and then exposed to DCQA for 24 h at a series of concentrations in LPS presence of 100 ng/mL. Subsequently, MTT solution was supplemented to determine the cell viability of DCQA. The optical density was measured using Bio-Rad microplate reader (CA, USA) at 570 nm.

The mRNA expression of pro-inflammatory cytokines by RT-qPCR
Total RNA was extracted and transcribed into cDNA according to the introduction of RT-qPCR reaction kit. The primer sequences (

Statistical analysis
Data were presented as mean ± SD. The survival percentage was determined by Chi-squared of exact test. Other statistical differences were carried out using the two-tailed Student's test by SPSS software (Chicago, USA). P < 0.05 was regarded as statistically significance.

Effects of DCQA on survival percentage of CLP mice
After CLP operation, only three mice were survived for 96 h in the control group with a survival percentage of 15 % (Figure 2). However, the survived mice after CLP were dosedependently increased by treatment with DCQA (7.5, 15, and 30 mg/kg), while the survival percentages were 35, 50, and 65 %. These findings suggested that treatment with DCQA markedly increased the survival percentage of CLP mice.

DCQA decreased LPS activity in vitro
As can be seen in Figure 4, LPS level was found to decrease following with co-incubation of DCQA (2, 4, and 8 μg/mL). These results indicated that DCQA has the capacity to neutralizing LPS level. MTT assay revealed that DCQA at 5, 10, 20, 40, 80, and 160 μg/mL showed an extremely low cell proliferation inhibition on RAW 264.7 cells treated with LPS ( Figure 5A). Furthermore, the RT-qPCR results ( Figure 5B-D) displayed that the TNF-α, IL-6, and IL-8 were gradually decreased after incubation with DCQA (10, 20, and 40 μg/mL). These findings suggested that the protective effects of DCQA against sepsis mice may be associated with the antiinflammatory property.

DISCUSSION
Sepsis is a serious condition for which an effective drug or treatment strategy is needed.
There is no doubt that compounds and extracts derived from traditional Chinese herbal medicine are beneficial for human health. Increasing number of studies suggested that some natural products isolated from herbal medicine exhibited protective effects against sepsis [15]. This is the first investigation to study the protective effects of DCQA against sepsis by CLP-induced mice model and LPS-treated RAW 264.7 cells.
An inflammatory reaction may be not only related to the innate immune response, but can also be associated with the pathogenesis of several conditions such as sepsis and cancer [16].
Recent studies have confirmed that some proinflammatory cytokines of TNF-α and IL-6 act essential role in sepsis development. It has been indicated that the TNF-α is one of the important mediators in the produce of inflammatory response syndrome, and IL-6 and IL-8 are the downstream cytokines [17]. After the inflammatory response is activated, numerous secondary inflammation mesons can be triggered which may ultimately cause multiple organ dysfunction.
An increasing number of investigations revealed that all three cytokines are related to the cytokine activation of cascade reaction in sepsis [18], and thus, suppression of the inflammatory response is critical in the treatment of sepsis. The results of this study indicated that DCQA can not only reduce TNF-α and IL-6 in sepsis mice induced by CLP surgery, but it can also inhibit the TNF-α, IL-6, and IL-8 expression in RAW 264.7 cells exposed to LPS.
Cecal ligation and puncture (CLP) is considered as a good experimental model to screen effective drugs for treating sepsis. It allows several intestinal bacteria to enter the abdominal cavity, followed by bacteria peritonitis and the transfer of bacteria into circulation [19]. The CLP surgery was applied to establish a pathological model of sepsis mice in the present study. On treatment with 7.5, 15, and 30 mg/kg of DCQA, the survival percentage of mice was increased to 35, 50, and 65 %, respectively. In addition, LPS is a trigger for sepsis and directly involved in the pathogenesis of a series of inflammatory response. Then, it activated immune reaction and damaged multiple organs [20]. RAW 264.7 cells treated with LPS were used for this investigation, and the TNF-α, IL-6, and IL-8 expression were decreased in all groups treated with DCQA (7.5, 15, and 30 mg/kg). Furthermore, the neutralization or inhibition of LPS would be beneficial in preventing severe sepsis, which can be viewed as a new therapeutic method [21]. The results in this study exhibited that DCQA had neutralizing effect on LPS in vitro.

CONCLUSION
The findings of this study, for the first time, show that 4, 5-O-dicaffeoylquinic acid (DCQA), a major phenylpropanoid derivative isolated from X. sibiricum, has the potential to be developed as an effective drug for the prevention of CLPinduced sepsis in mice. It acts by decreasing TNF-α, IL-6, and IL-8. This investigation provides a fresh insight into the medicinal potentials of the bioactive constituents of X. sibiricum.