The Research Of Inflammatory Signaling Pathways Of Xijiao Dihuang Decoction Treating Ischemic Stroke In Vitro

PurposesWe plan to explore whether Xijiao Dihuang decoction(aliased as XJDH decoction)can protect neuron from ischemia-reperfusion injury and reduce the damage caused by inflammation in vitro experiments.Further,we expect to clarify whether TLR4/MyD88 signaling pathway associated with inflammation plays an important role in mechanisms of XJDH decoction treating ischemic stroke.Through this research,we hope to deepen the theory of Yure of TCM in the treatment of ischemic stroke,and provide a new strategy for the therapy of ischemic stroke.MethodsIn this study,we conduct our experiments in vitro cells with oxygen-glucose deprivation and reoxygenation which is a vitro model of cerebral ischemia and reperfusion.Cells were divided into four groups:vehicle control,OGD/R,OGD/R +XJDH,and OGD/R+paeoniflorin.For the vehicle control group,PC 12 cells were not exposed to OGD/R.For the OGD/R group,the original medium was removed,and then cells were rinsed with PBS(0.01 M,pH 7.4)twice,and exposed to DMEM(glucose free)and cultured in a sealed anaerobic chamber flushed with 5%CO2 and 95%N2(v/v)for 6 h.XJDH and paeoniflorin were respectively added at 30 min before OGD exposure.Then,a reoxygenation period(24 h)was begun by rapidly replacing the DMEM(glucose free)with normal medium and conditions.We decide to choose PC 12 to complete our study.First of all,the contents of three major compounds in XJDH decoction are evaluated by high performance liquid chromatography(HPLC).Secondly,according to MTT test and LDH release assay,we try to detect the effect of XJDH and paeoniflorin on cell toxicity and the viability of cells induced by OGD/R.The level of inflammatory factors(containing IL-6,IL-1 and TNF-alpha)and the concentration of intracellular caspase-3 are determined by ELISA in order to understand the effective mechanism of XJDH in respect of inflammation and apoptosis-The Western blot assay is used to measure the protein expression levels of TLR4,MyD88,NF-kappa B,TRAF6,p-ERK,p-JNK,Akt and caspase-3 in cells,so as to make sure the possible signal path of drug intervention.ResultsThe results show that the contents of paeoniflorin,albiflorin,and oxypaeoniflorin in XJDH decoction are 0.232±0.031%,0.276±0.018%,and 0.019±0.002%,respectively.With PC2 cells,it was found that OGD/R significantly decreased the cell viability and LDH activity,and increased the release of IL-1β,IL-6,TNF-α in PC12 cells;these effects were suppressed by XJDH and one of its major active constituents,paeoniflorin.XJDH also inhibited activity of caspase-3 and inhibited the level of cleaved caspase-3.Mechanistic studies showed that XJDH attenuated the OGD/R-induced increases in TLR4,MyD88,TRAF6,and NF-κB,and secretion of TNF-α,IL-6,IL-1β.However,XJDH did not reversed the OGD/R-induced increases in p-JNK,p-ERK1/2 and p-p38 expression.The results showed that the neuroprotective effect of XJDH was carried out mainly by regulating the TLR4/MyD88/NF-κB signaling pathways.As an anti-inflammation factor,XJDH might be used as a neuronal protection strategy for the ischemia injury and related diseases.Conclusions1.XJDH decoction can lighten the OGD/R damage in PC 12 cells to protect neuron.2.XJDH decoction can decrease inflammatory factor levels by inhibiting TLR4/MyD88 signaling pathway and cell apoptosis to show its neural protective function.3.The theory of Yure of TCM is valuable in the study of ischemic stroke,and XJDH decoction is expected to provide a new way of treatment for ischemic stroke.