Study On The Mechanism Of Epimedin B Against LPS-induced Inflammatory Response In Primary Astrocytes

Neuroinflammation is gradually regarded as an important pathological mechanism of neurodegenerative diseases.The secretion of inflammatory cytokines from microglia and astrocytes will seriously damage the surrounding neurons.Inhibiting excessive activation of glial cells may be an important target for the treatment of neurodegenerative diseases.Astrocytes originate from the neuroectoderm and are widely distributed in the brain.On the one hand,astrocytes play an important role in the nutritional support of neurons by releasing various neurotrophic factors.On the other hand,under pathological conditions,increasing number of reactive astrocytes can trigger an inflammatory response and eventually damage neurons.Total flavonoid of Herba Epimedii are flavonoids extracted from the leaves of Epimedium,which mainly contain icariin and Epimedin B et al.Modern pharmacological studies have found that total flavonoid of Herba Epimedii are the effective components of Epimedium in promoting immune function,participating in bone metabolism,anti-tumor,enhancing cardiovascular activity and exerting the neuroprotective function.Epimedin B is the main active compound of the total flavonoid of Herba Epimedii,and its content is second only to icariin.A recent study had found that Epimedin B could stimulate the proliferation and differentiation of rat UMR106 cells,and this effect could be blocked by the specific antagonist of nuclear estrogen receptor(ER)ICI182,780.However,the ligand binding assay revealed that Epimedin B could not directly bind to ER,suggesting that ER may be activated through other signaling pathways.G protein-coupled estrogen receptor(GPER)is a new type of membrane estrogen receptor.Estrogen can exert anti-inflammatory and anti-apoptotic effects through the GPER-mediated rapid non-genomic mechanism.Our previous work proved that the total flavonoid of Herba Epimedii and its main active compound icariin could inhibit LPS-induced astrocyte inflammation.Epimedin B,as the second highest active compound in the total flavonoid of Herba Epimedii,if it can also inhibit the inflammatory response of astrocytes? And,what is the mechanism of its anti-inflammatory effects? So far,there have been few studies on the above questions.Objective: 1.To investigate the inhibitory effect of Epimedin B on the inflammation of primary astrocytes and its mechanism.2.To study the regulatory effects of Epimedin B on the physiological functions of primary astrocytes.Methods: 1.LPS was used to induce the inflammatory model of rat primary astrocyte.Real-time PCR and Western blot were used to determine the mRNA and protein expressions of inflammatory cytokines.Silencing GPER by siRNA interference technology was used to observe the anti-inflammatory effect of Epimedin B.2.Astrocytes were treated with Epimedin B alone,and then Real time PCR technology was used to detect its effect on the gene expressions of c-fos,GLAST and GLT-1.Results: 1.Study of the inhibitory effects of Epimedin B on the inflammatory response of astrocytes(1)In order to determine the purity of the primary cultured astrocytes,immunofluorescence technology was used to detect the expression of GFAP in primary astrocytes.The results showed that more than 95% of the cells were astrocytes.(2)LPS was used to treat astrocyte for 6 h,the mRNA expressions of TNF-?,IL-1?,iNOS and COX-2 were significantly up-regulated(P<0.001).Pretreatment with different doses of Epimedin B(1,10 and 20 ?M)could inhibit the LPS-induced inflammatory response in a dose-dependent manner.Epimedin B at a concentration of 10 ?M works best(P<0.05),so it will be used for subsequent experiments.(3)GPER-specific antagonist G15(1 ?M)could block the inhibitory effects of Epimedin B on LPS-induced mRNA expressions of TNF-?,IL-1?,COX-2,and the difference is statistically significant(P<0.01,P<0.05,P<0.01).But it had no blocking effect on iNOS mRNA expression.ER-specific antagonist ICI182,780 could not significantly block the inhibitory effect of Epimedin B on LPS-induced inflammatory factors gene expressions.(4)G15 and ICI182,780 could significantly block the inhibitory effect of Epimedin B on LPS-induced COX-2 protein expression(P<0.05),and had a partly blocking effect on iNOS expression while the difference is not statistically significant(P>0.05).(5)SiRNA obviously silenced the mRNA and protein expression of GPER in primary astrocytes(P<0.001,P<0.01).Compared with the negative control group,the knockdown rates were 60% and 50%,respectively.(6)After silencing GPER gene expression with siRNA,the inhibitory effects of Epimedin B on LPS-induced up-regulation of TNF-?,IL-1?,and COX-2 gene level were significantly antagonized(P<0.05).(7)After silencing GPER gene expression with siRNA,the inhibitory effect of Epimedin B on LPS-induced up-regulation of COX-2 protein level was also significantly antagonized(P<0.05).2.Study of the regulatory effect of Epimedin B on the physiolgical functions of astrocytes(1)Epimedin B was used to treat astrocytes alone for 1,2,3 or 4 h.Real time-PCR results showed that the gene expression of c-fos increased at 1 h(P<0.01),and the difference was statistically significant(P<0.05),suggesting that Epimedin B can activate astrocytes.(2)The mRNA expression of GLAST was significantly up-regulated when treated with Epimedin B for 3 h(P<0.05),and the mRNA expression of GLT-1 was significantly up-regulated when treated with Epimedin B for 4 h(P<0.05),suggesting that Epimedin B can regulate the physiological functions of astrocytes.Conclusion:The experimental results show that Epimedin B can inhibit LPS-induced astrocyte inflammatory response and estrogen membrane receptor GPER may be the major target.Epimedin B treatment alone can regulate the physiological functions of astrocytes.It is of great significance to maintain the homeostasis of the surrounding environment of astrocytes.