Elsevier

Journal of Ethnopharmacology

Volume 185, 5 June 2016, Pages 77-86
Journal of Ethnopharmacology

Geniposide attenuates inflammatory response by suppressing P2Y14 receptor and downstream ERK1/2 signaling pathway in oxygen and glucose deprivation-induced brain microvascular endothelial cells

https://doi.org/10.1016/j.jep.2016.03.025Get rights and content

Abstract

Ethnopharmacological relevance

Fructus gardenia is widely used for treatment of stroke and infectious diseases in Chinese medicine. Geniposide is the key bioactive compound related to the pharmacodynamic actions of gardenia on ischemic stroke. The molecular mechanism by which geniposide improves the ischemic brain injury was observed in the study.

Aim of the study

Recent studies showed that geniposide had protective activities against the inflammatory response in ischemic stroke. However, the molecular mechanism of geniposide anti-inflammatory role has not yet been fully elucidated. In this study, we investigated the effect of geniposide on the expression of P2Y14 receptor and downstream signaling pathway in brain microvascular endothelial cells (BMECs).

Materials and methods

An in vitro model of cerebral ischemia in BMECs was established by oxygen-glucose-deprivation (OGD). To further confirm the specific effect of geniposide on P2Y14 receptor and downstream signaling pathways, we set up a UDP-glucose (an agonist of the P2Y14 receptor) stimulated model. After administration of geniposide, the expression of P2Y14 receptor, phosphorylation of RAF-1, mitogen activated protein kinase kinase1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), level of interleukin-8 (IL-8), interleukin-1β (IL-1β), monocyte chemotactic protein 1 (MCP-1) in BMECs were determined.

Results

The mRNA and protein expression of P2Y14 in the rat BMECs were up-regulated in OGD-induced injury. After administration of Geniposide, the expression of P2Y14 receptor was significantly down-regulated, the phosphorylation of RAF-1, MEK1/2, ERK1/2 were suppressed. Similar data were obtained in UDP-glc stimulated model. We also observed that geniposide markedly declined the production of IL-8, IL-1β and MCP-1 in OGD-induced BMECs.

Conclusion

Geniposide exerted anti-inflammatory effects by interfering with the expression of P2Y14 receptor, which subsequently inhibits the downstream ERK1/2 signaling pathways and the release of the pro-inflammatory cytokines IL-8, MCP-1, IL-1β. Therefore, this study provides the evidence for gardenia's clinical application in cerebral ischemia.

Introduction

Cerebral ischemia is a result of reduced cerebral blood flow to a discrete region of the brain, and this initiates a complex process in which the inflammatory response has been recognized as one of the main pathological factors in the development of brain injury. The brain microvascular endothelial cells (BMECs), as a highly specialized endothelial tissue that performs the function of Blood-brain barrier, appears to be an important responsive and regulatory component of cerebral inflammation and a primary target of immunological attack by oxygen-glucose-deprivation (OGD) (Stanimirovic and Satoh, 2000). The activation of BMECs triggers excessive production of pro-inflammatory cytokines, including various chemokine and adhesion factors, which further recruits neutrophils to endothelium spaces and amplify the inflammatory response by subsequent release of more inflammatory mediators (Jean et al., 1998, Zhang et al., 2013a, Zhang et al., 2013b). Therefore, targeting activated BMECs and related factors could be a crucial therapeutic strategy for attenuating the inflammatory response and preventing brain damage in cerebral ischemia.

P2Y purinergic receptors, as seven-spanning G protein-coupled receptors, were gradually realized to play an important role in the peripheral and neuroimmune function in the past decade. So far, eight human subtypes of the P2Y receptor family have been defined, respectively P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 (Harden et al., 2010), which modulate a number of diverse physiological process in response to extracellular nucleotides, nucleosides or nucleotides sugars (la Sala et al., 2003). P2Y14 receptor, identified initially as a receptor activated by UDP-glucose and other UDP-sugars, is the newest member in this family. Although P2Y14 receptor has a relatively broadly expression in the body, and has been found in the brain, lung, spleen, heart, gastrointestinal smooth muscle, hematopoietic stem cell, and many types of immune cell, the signaling properties and physiological roles of P2Y14 receptor remain very limited (Moore et al., 2003, Chambers et al., 2000, Scrivens and Dickenson, 2005, Chootip et al., 2005). Recent studies indicated that P2Y14 receptor played a role in the regulation of immune responses, including chemotaxis of bone-marrow hematopoietic stem cells, cytokine IL-8 production, inhibition of T-lymphocytes and neutrophils proliferation (Scrivens and Dickenson, 2005, Scrivens and Dickenson, 2006, Xu et al., 2012, Lee et al., 2003, Muller et al., 2005). It was reported that P2Y14 receptor mRNA was up-regulated in several brain regions after immunological challenge of mice with lipopolysaccharide (Moore et al., 2003). However, at present it is not known whether the P2Y14 receptor is functionally expressed in cerebral endothelial cells and whether P2Y14 receptor is involved in inflammatory responses during ischemic stroke. Therefore, in the present study we investigated the expression and signaling properties of P2Y14 receptor in in vitro mock ischemic BMECs.

Fructus gardenia (called Zhizi in Chinese), derived from the dried fruit of Gardenia jasminoides Ellis, is one of the most popular traditional herbs in treatment of ischemic stroke according to the theory of traditional Chinese medicine (Xiao et al., 2011, Ai et al., 2012). The iridoid glycosides is the characteristic constituents of Gardenia, among them geniposide constitutes the highest proportion and is often chosen to be the marker component for the quality control of Gardenia. The comparison of the activity of the plant extract and geniposide had been done, and found geniposide was the main compound in extract for anti-inflammatory and anti-thrombotic activity (Koo et al., 2006, Zhang et al., 2013a, Zhang et al., 2013b). Besides, some reports showed that geniposide had effects of antioxidation, anti-inflammatory, anti-ischemic brain injuries, anti-platelet aggregation (Liu et al., 2013), which demonstrated the geniposide is the key bioactive compound related to the pharmacodynamic actions of Gardenia on ischemic stroke. However, the molecular mechanism of geniposide anti-inflammation in ischemia is unclear, especially there are no data about the effect of geniposide on P2Y14 receptor expression in BMECs exposed to OGD. Herein, we assessed the effect of geniposide on P2Y14 receptor expression and the underlying mechanism of action, which would offer pharmacological proof for Gardenia's clinical application in cerebral ischemia.

Section snippets

Materials

Uridine 5′-diphospho glucose (UDP-glucose) and pertussis toxin (PTX) were purchased from Sigma Aldrich (St. Louis, MO, USA). Rabbit anti-rat P2Y14 antibody was purchased from Alomone Labs (Har Hotzvim, Jerusalem). Rabbit anti-rat p-Raf, p-MEK1/2, p-ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). ELISA kits for rat interleukin-8 (IL-8), interleukin-1β (IL-1β), monocyte chemotactic protein 1(MCP-1) were purchased from R&D Systems (Minneapolis, MN, USA).

P2Y14 receptor was expressed in rat BMECs and up-regulated by exposure to OGD in mRNA and protein level

Expression of the P2Y14 receptor in normal and OGD-injured BMEC was analyzed using real-time PCR and western blotting. As shown in Fig. 2A, the expression of P2Y14 mRNA was detected in normal rat BMECs and was increased in OGD group (P<0.01). Fig. 2B and C showed the result of the P2Y14 protein level by western blot, which was consistent with real-time PCR. These results collectively suggested P2Y14 receptor was expressed in rat BMECs and could be obviously up-regulated by exposure to OGD in

Discussion

Purinergic receptors, including P1 adenosine receptor, P2X ionotropic receptor and P2Y metabotropic receptor, are a family of transmembrane proteins that is activated by nucleosides, nucleotides, and nucleotide sugars. Recent studies showed that purinergic signaling had a pivotal role in alerting and tuning immune and inflammatory reactions to aversive influences in the central nerve system (Di Virgilio, et al., 2009). Brain damage can cause release of nucleotides and nucleotides sugars–which

Conclusions

Taken together, here we provide the evidence of P2Y14 receptor expression in rat cerebral microvascular endothelial cell. Our results support the hypothesis that: 1) P2Y14 receptor and downstream ERK1/2 signaling pathways are activated in OGD-injured BMECs, which might contribute to post-ischemia inflammatory reactions. 2) Geniposide exerts anti-inflammatory effects by interfering with the expression of P2Y14 receptor, which subsequently inhibits the downstream ERK1/2 signaling pathways and the

Disclosure/conflict of interest

None.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81273885), the 111 project (Grant no. B07007), “Nautical Traditional Chinese Medicine” Collaborative Innovation Center (No. 522/0100604299).

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    Bingce Wang, contributing to analysis of the data and process of picture.

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    Guangrui Huang, guiding the experimental methods.

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