| Intracerebral hemorrhage(ICH)refers to primary intracerebral hemorrhage with high incidence rate,mortality and disability.Primary brain injury(PBI)and secondary brain injury(SBI)after ICH are the main causes of high mortality and disability.Inflammatory reaction and apoptosis play an important role in SBI after ICH.Studies have shown that microglia display pro-inflammatory phenotype(M1)and anti-inflammatory phenotype(M2)after being activated around ICH lesions,and promote the transformation of microglia phenotype into M2 type,which can promote its anti-inflammatory and anti-apoptotic effects.Peroxisome proliferator-activated receptorγ(PPARγ)is a transcription factor.ICH in vivo and in vitro experiments found the activation of PPARγ can promote the transformation of microglia around the hematoma into M2 type,reduce the nerve function defect after rats with ICH,and PPARγ Inhibitor GW9662 can eliminate protective effect.Therefore,regulating the transformation of microglia phenotype into M2 type through PPARγ is an important intervention target for the treatment of ICH.ICH belongs to the category of "stroke" in traditional Chinese medicine,which is often called "hemorrhagic stroke" in modern times to distinguish ‘ischemic stroke’.The treatment of hemorrhagic stroke with traditional Chinese medicine has been widely valued.The basic pathogenesis of hemorrhagics troke is the imbalance of Yin and Yang and the disorder of Qi and blood,resulting abnormal rising of chong-qi and the overflow of blood from arteries of brain.According to this pathogenesis,the An-nao-ping-chong decoction,originated by neurology department from the first hospital of Hunan University of Chinese Medicine,is the agreement decoction for the treatment of hemorrhagic stroke.It’s main takes main treatment method is Pingchong Jiangni,and has been widely used in clinical practice for many years,with significant clinical effects,but itsexperimental level research and molecular mechanism research are still lacking.The main purpose of this study is to explore whether the serum containing An-nao-ping-chong decoction can regulate the phenotype of microglia and its possible molecular mechanism at the level of in vitro research;Secondly,we further verified from the in vivo experiment whether the An-nao-ping-chong decoction can reduce the SBI after rats with ICH and the possible molecular mechanism,and provide new basis and new idea for the treatment of ICH with An-nao-ping-chong decoction,meanwhile provide new ideas for the treatment of ICH with traditional Chinese medicine.Part Ⅰ In VitroObjective: To explore the protective effect of serum containing An-nao-ping-chong decoction(A-HYXQ)on BV2 microglia injured by Ferriheme chloride(Hemin)Methods: BV2 microglia were injured by Hemin,and the model of inflammatory reaction after ICH was established in vitro.After screening the optimal concentration of Hemin and A-HYXQ,the experiment was divided into three groups: control group,model group and A-HYXQ group.Immunofluorescence(IF)and Western blot(WB)were used to detect the expression level of chitinase-like protein 3(Ym1)and Interleukin-10(IL-10),the markers of M2 microglia,and the expression level of inducible nitric oxide synthase(i NOS)and Interleukin-6(IL-6),the markers of M1 microglia.Results: Compared with the control group,the expression level of M2 microglial markers(Ym1 and IL-10)in the model group decreased significantly(P<0.05),while the expression level of M1 microglial markers(i NOS and IL-6)increased significantly(P<0.05);Compared with the model group,A-HYXQ group could significantly reduce the expression level of i NOS and IL-6(P<0.05),and significantly increase the expression level of Ym1 and IL-10(P<0.05).Conclusion: A-HYXQ can reduce the inflammatory reaction of BV2 microglia injuried by Hemin by regulating the transformation of microglia phenotype into M2 type.Part Ⅱ In VitroObjective: To explore the molecular mechanism of the phenotypic regulation of A-HYXQ on BV2 microglia injuried by Hemin.Methods: BV2 microglia were injured by Hemin,and the model of inflammatory reaction after ICH was established in vitro.Screening the best concentration of GW9662,PPARγ inhibitor.GW1929,agonist of PPARγ was used as the positive control drug.The experiment was divided into five groups: control group,model group,A-HYXQ group,A-HYXQ+GW9662 group,and GW1929 group.Detection the expression level of M2 microglial marker,PPARγ,arginase-1(Arg-1)and Interleukin-4(IL-4),by IF,WB and real-time fluorescence quantitative polymerase chain reaction(q RT-PCR);Detection the expression level of M1 microglial marker,tumor necrosis factor-α(TNF-α)and Interleukin-1 β(IL-1β)by WB,q RT-PCR and enzyme-linked immunosorbent assay(ELISA).Results: Compared with the control group,the expression level of PPARγ,Arg-1and IL-4 decreased significantly(P<0.05)in the model group,while the expression level of TNF-α and IL-1βwas significantly increased(P<0.05)in the model group;Compared with model group,A-HYXQ group and GW1929 group can significantly reduce the expression of TNF-α and IL-1β,and significantly increased the expression level of PPARγ,Arg-1 and IL-4(P<0.05);Compared with A-HYXQ group,A-HYXQ+GW9662 group dramatically decreased in the expression of PPARγ,Arg-1 and IL-4(P<0.05)while the expression level of TNF-α and IL-1β increased significantly(P<0.05);Compared with A-HYXQ group,the expression of PPARγ and Arg-1 increased more significantly(P<0.05)in GW1929 group,meanwhile,the decrease in the expression of IL-1β was more obvious(P<0.05),but the expression of IL-4 and TNF-α no significant statistical difference(P>0.05).Conclusion:1.Activating PPARγ signal pathway can regulate the transformation of microglia phenotype into M2 type,reducing the inflammatory reaction of BV2 microglia damaged by Hemin.2.A-HYXQ can partially activate PPARγ signal pathway regulates the transformation of microglia phenotype into M2 type,reducing the inflammatory response of BV2 microglia injured by Hemin.Part Ⅲ In VivoObjective: Based on PPARγ signal pathway polarizes M2 microglia,and discusses the effect and mechanism of An-nao-ping-chong decoction on reducing SBI after rats with ICH.Methods: The ICH model in vivo was established by injecting autologous arterial blood into the left basal ganglia of rats.Male Sprague Dawley(SD)rats were randomly divided into four groups: sham operation group,model group,An-nao-ping chong-fang group,An-nao-ping chong-fang+GW9662 group.The nerve function defect of rats after ICH was evaluated by using the modified neurological severity score(m NSS),forelimb placement test and corner test at three different time points,1d,3d and 7d,after ICH;Hematoxylin-eosin(HE)staining was used to observe the brain tissue structure and inflammatory cell infiltration around the hematoma;The water content of the brain tissue was measured by dry/wet method;the apoptotic cells in the brain tissue around the hematoma were observed by terminal deoxynucleotidyl transferase mediated d UTP nick-end labeling(TUNEL)staining,and the apoptotic rate was calculated.The apoptosis-related markers,B-cell lymphoma-2(Bcl-2),BCL2 associated X protein(Bax)and caspase-3,in the brain tissue around the hematoma was detected by WB and q RT-PCR;the expression level around hematoma of M2 microglial cell marker(PPARγ,Arg-1 and IL-4)and M1 microglial cell marker(TNF-α and IL-1β)were detected by IF,WB,q RT-PCR and ELISA.Results: Compared with the control group,the m NSS score increased significantly(P<0.05),and the success rate of placing the right forelimb and the odds of turning right decreased significantly(P<0.05);inflammatory cell infiltration in the brain tissue around the hematoma increased significantly,and the number of apoptotic cells around the hematoma and brain water content increased significantly(P<0.05);and the expression of Bcl-2,PPARγ,Arg-1and IL-4 around the hematoma decreased significantly(P<0.05);while the expression of Bax,Caspase-3,TNF-α and IL-1β around the hematoma increased significantly(P<0.05)in the model group.Compared with the model group,the m NSS score decreased significantly(P<0.05),and the success rate of placing the right forelimb and the odds of turning right increased significantly(P<0.05);inflammatory cell infiltration in the brain tissue around the hematoma decreasedsignificantly,and the number of apoptotic cells around the hematoma and brain water content decreased significantly(P<0.05);and the expression of Bcl-2,PPARγ,Arg-1 and IL-4 around the hematoma increased significantly(P<0.05);while the expression of Bax,Caspase-3,TNF-α and IL-1β around the hematoma decreased significantly(P<0.05)in the An-nao-ping-chong group;Compared with the An-nao-ping-chong group,the m NSS score increased significantly(P<0.05),and the success rate of placing the right forelimb and the odds of turning right decreased significantly(P<0.05);inflammatory cell infiltration in the brain tissue around the hematoma increased significantly,and the number of apoptotic cells around the hematoma and brain water content increased significantly(P<0.05);and the expression of Bcl-2,PPARγ,Arg-1 and IL-4around the hematoma decreased significantly(P<0.05);while the expression of Bax,Caspase-3,TNF-α and IL-1β around the hematoma increased significantly(P<0.05)in the An-nao-ping-chong+GW9662 group.Conclusion: An-nao-ping-chong decoction can partially activate PPARγ signal pathway regulates the transformation of microglial cell phenotype into M2 type,reduceing the inflammatory reaction and cell apoptosis after rats with ICH,decreasing brain edema.Therefore,reducesing the SBI after rats with ICH,and finally achieves the goal of improving the prognosis of neural function in rats with ICH. |
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