The Mechanisms Of Post-ischemic Neuroinflam Mation Regulated By Ramulus Cinnamomi-Herba Ephedrae Based On TLR4/MyD88/MAPK Pathway

Background:Recently,cerebral ischemia has been considered to be one of the most common symptoms in cerebrovascular diseases,which is one of the diseases with the highest mortality rate.It has been reported that an increase in the levels of inflammatory factors—such as tumor necrosis factor-alpha(TNF-?),interleukin-6(IL6)and interleukin-1 beta(IL-1?)—are likely to be one of the most important causes of cerebral ischemia.It has also been reported that the Ramulus Cinnamomi-Herba Ephedrae can inhibit the activation of those inflammatory factors.However,the mechanism of post-ischemic neuroinflammation regulated by Ramulus Cinnamomi-Herba Ephedrae is still unknown and some essential experiments are needed to elucidate it.Objective:This research was designed to evaluate the effect of traditional Chinese medicine Ramulus Cinnamomi-Herba Ephedrae for the treatment of cerebral ischemia in rats,and investigate the inhibitory effect of Ramulus Cinnamomi-Herba Ephedrae on the neuroinflammation in the ischemic stroke through TLR4/MyD88/MAPK pathway,as well as its underlying mechanisms in vivo and vitro.Methods:(1)A cerebral ischemic-reperfusion injury(CIRI)model was established by using pulsinelli four-vessel occlusion(4-VO).After the model was successfully established,the rats were given Ramulus Cinnamomi-Herba Ephedrae(Ramulus Cinnamomi:Herba Ephedrae=2:3;3,6 and 12g/kg,i.g.)by intragastric administration for 7 days,then observed the changes between the different groups.The light and dark shuttle box test and the Morris water maze test were used to evaluate the rats' memory behavior.(2)The prefrontal cortex and hippocampus of the rats were extracted,Hematoxylin-Eosin(H-E)was used for histopathological observation.(3)The expression levels of TNF-?,IL6,and IL-1? in pro-inflammatory cells were detected by enzyme-linked immunosorbent assay(ELISA).(4)The expression levels of TLR4,MyD88,MAPK(p38,ERK,JNK)in rats' cortex and hippocampus were detected by RT-PCR and Western Blotting method before and after the intervention.(5)In vitro study,LPS-induced inflammatory models were established by primary glial cells,before treating with Ramulus Cinnamomi-Herba Ephedrae.The concentrations of TNF-a and IL-6 in the supernatant of each group were detected by corresponding ELISA kits.The RT-PCR and Western Blotting methods were used for Ramulus Cinnamomi-Herba Ephedrae detection,in order to explore the effects of the key genes and proteins associated with inflammation in this test.Results:(1)The effect of Ramulus Cinnamomi-Herba Ephedrae on learning and memory functions of the CERI model rats.Firstly,passive avoidance test showed that the latency time at 24 h after initial training was reduced,whereas the error time was increased after the surgery.However,both of the above parameters were reversed by Ramulus Cinnamomi-Herba Ephedrae(6 and 12 g/kg,i.g.)without affecting the latency during the training session.Secondly,Morris water maze test showed that the swimming distance in platform quadrant was decreased in cerebral ischemia animals,as well as the duration period decreation in target quadrant,respectively.However,both of the results were reversed by Ramulus Cinnamomi-Herba Ephedrae(6 and 12 g/kg,i.g.).Finally,the animal beha'vior experiments showed that the Ramulus Cinnamomi-Herba Ephedrae(6 and 12 g/kg,i.g.)significantly improves the symptoms in memory-related behavioral disorders caused by cerebral ischemia.(2)The effect of Ramulus Cinnamomi-Herba Ephedrae on brain tissue structure of the CIRI model rats.HE strain test provided a credible evidence that Ramulus Cinnamomi-Herba Ephedrae(6,and 12 g/kg i.g.)can improve the pathological phenomenons in stuctural damage models induced by ischemia,with the performances that the number of cells increased,cellular stuctures were more intact,the number of abnormal cells were fewer.We can conclude that Ramulus Cinnamomi-Herba Ephedrae can alleviate cerebral injury.(3)The effect of Ramulus Cinnamomi-Herba Ephedrae on the expression levels of TNF-?,IL-6 and IL-1? in brains.ELISA analysis showed that the levels of TNrF-a in the prefrontal cortex and hippocampus in this models were significantly increased(P<0,05).After the intervention by Ramulus Cinnamomi-Herba Ephedrae(6 and 12 g/kg,P<0.05),TNF-? levels were significantly reduced.Meanwhile,the expression levels of IL-6 and IL-1? were significantly increased after modeling(P<0.05).All of them were significantly decreased after treatment with Ramulus Cinnamomi-Herba Ephedrae,which were increased significantly before the intervention.It was concluded that the Ramulus Cinnamomi-Herba Ephedrae(6 and 12 g/kg,i.g.)can significantly inhibit the increase of TNF-a,IL-6 and IL-1p levels.(4)The effect of Ramulus Cinnamomi-Herba Ephedrae on the inflammatory pathways.In our tests,compared to the control group,the downregulations of protein expressions of TLR4,MyD88 were observed in the Ramulus Cinnamomi-Herba Ephedrae groups.Besides,the phosphorylation levels of p38,JNK and ERK were influenced significantly by Ramulus Cinnamomi-Herba Ephedrae.RT-PCR tests showed that the downregulation of gene expression of TLR4,MyD88 were obvious in Ramulus Cinnamomi-Herba Ephedrae group.The high dose of Ramulus Cinnamomi-Herba Ephedrae treatment(12 g/kg,i.g.)can improve the pathological phenomenon compared to the normal animals.(5)The inhibitory effect of Ramulus Cinnamomi-Herba Ephedrae on inflammation and TLR4/MyD88/MAPK inflammatory pathway.ELISA analysis showed the TNF-?,IL-6 levels were significantly increased in the cell supernatant of the model group.This levels were decreased after the Ramulus Cinnamomi-Herba Ephedrae intervention,in which the high dose of Ramulus Cinnamomi-Herba Ephedrae(12 g/kg,i.g.)can inhibit the secretion of inflammatroy factors.Our test also demonstrated that Ramulus Cinnamomi-Herba Ephedrae can inhibit the gene and protein expression of TLR4,MyD88.Besides,the phosphorylation levels of p38,JNK and ERK were affected by Ramulus Cinnamomi-Herba Ephedrae.Conclusion:(1)Ramulus Cinnamomi-Herba Ephedrae have the positive effects on the improvement of the learning and memory ability of CIRI model rats.(2)Ramulus Cinnamomi-Herba Ephedrae have the positive effects on the alleviation of the pathological damage of brain tissue in CIRI rat model.(3)Ramulus Cinnamomi-Herba Ephedrae can inhibits the inflammatory processes by down-regulating the expression levels of pro-inflanmmatory cytokines in the CIRI model rats,such as TNF-?,IL-6,and IL-1?.The mechanism may be associated to the inhibition of TLR4/MyD88/MAPK inflammatory pathways.(4)Ramulus Cinnamomi-Herba Ephedrae exhibit biologically activities at microglia and astrocyte,reducing the release of TNF-a and IL-6 by regulating TLR4-mediated inflammatory pathways,inhibiting neuroinflammation,through which neurons are protected from inflammation.