加味通络方含药血清调控JNK信号通路改善大鼠原代皮层神经元氧糖剥夺/复氧损伤的作用机制OA

Objective To investigate the mechanism of Jiawei Tongluo Formula-containing serum on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced injury in primary rat cortical neurons based on the c-Jun N-terminal kinase(JNK)signaling pathway.Methods Primary cortical neurons were isolated and cultured from fetal rats.Jiawei Tongluo Formula-containing serum and blank serum were prepared from rats.The OGD/R cell model was established by subjecting neurons to OGD for 6 hours followed by reoxygenation for 12 hours or 24 hours.Primary rat cortical neurons were randomly divided into the following groups:blank control,medicated serum,OGD6 h/R12 h,OGD6 h/R12 h+medicated serum,OGD6 h/R12 h+inhibitor,OGD6 h/R12 h+combination,OGD6 h/R24 h,and OGD6 h/R24 h+medicated serum.One hour prior to OGD treatment,the blank control group received 10%blank serum;the medicated serum group received 10%Jiawei Tongluo Formula-containing serum;the inhibitor group received 20 µmol·L-1 SP600125(JNK pathway inhibitor,dissolved in DMSO,final concentration＜0.1%);and the combination group received 10%medicated serum+20 µmol·L-1 SP600125.Immunocytochemistry/immunofluorescence(ICC-IF)was used to detect Nestin expression levels;CCK-8 assay was used to measure cell proliferation rate;Annexin V/PI double staining was used to detect apoptosis rate;qRT-PCR was used to detect the mRNA expression levels of MAPK8,JUN,Bcl-2,Fas,and Caspase-3;Western Blot was used to detect the protein expression levels of JNK,p-JNK,c-Jun,Fas,Cleaved-Caspase-3,and Bcl-2.Results(1)Compared with the blank control group,the medicated serum group showed significantly increased Nestin fluorescence intensity and cell proliferation rate(P＜0.05,P＜0.01),with no significant change in apoptosis rate(P＞0.05);the OGD6 h/R12 h and OGD6 h/R24 h groups exhibited significantly decreased Nestin fluorescence intensity and cell proliferation rate(P＜0.01),and significantly increased apoptosis rate(P＜0.01);the OGD6 h/R12 h group showed significantly upregulated protein expression of p-JNK,c-Jun,Fas,and Cleaved-Caspase-3(P＜0.01),significantly downregulated Bcl-2 protein expression(P＜0.01),and significantly upregulated mRNA expression of MAPK8,JUN,Fas,and Caspase-3(P＜0.01),along with significantly downregulated Bcl-2 mRNA expression(P＜0.01).(2)Compared with the model group at the same time point,the OGD6 h/R12 h+medicated serum,OGD6 h/R12 h+inhibitor,and OGD6 h/R24 h+medicated serum groups showed significantly increased Nestin fluorescence intensity and cell proliferation rate(P＜0.01),and significantly decreased apoptosis rate(P＜0.01);the OGD6 h/R12 h+medicated serum and OGD6 h/R12 h+inhibitor groups exhibited significantly downregulated protein expression of p-JNK,c-Jun,Fas,and Cleaved-Caspase-3(P＜0.01),significantly upregulated Bcl-2 protein expression(P＜0.01),and significantly downregulated mRNA expression of MAPK8,JUN,and Fas(P＜0.01),along with significantly upregulated Bcl-2 mRNA expression(P＜0.01);the OGD6 h/R12 h+medicated serum group showed significantly upregulated Caspase-3 mRNA expression(P＜0.05),while the OGD6 h/R12 h+inhibitor group exhibited significantly downregulated Caspase-3 mRNA expression(P＜0.01).(3)Compared with either the OGD6 h/R12 h+medicated serum group or the OGD6 h/R12 h+inhibitor group,the OGD6 h/R12 h+combination group showed significantly increased Nestin fluorescence intensity and cell proliferation rate(P＜0.01),and significantly decreased apoptosis rate(P＜0.01);protein expression of p-JNK,c-Jun,Fas,and Cleaved-Caspase-3 was significantly downregulated(P＜0.01),Bcl-2 protein expression was significantly upregulated(P＜0.01),and mRNA expression of MAPK8,JUN,Fas,and Caspase-3 was significantly downregulated(P＜0.01),while Bcl-2 mRNA expression was significantly upregulated(P＜0.01).(4)Compared with the OGD6 h/R12 h group,the OGD6 h/R24 h group showed no significant differences in Nestin fluorescence intensity or cell proliferation rate(P＞0.05);however,compared with the OGD6 h/R12 h+medicated serum group,the OGD6 h/R24 h+medicated serum group exhibited a significantly decreased cell proliferation rate(P＜0.01).Conclusion Jiawei Tongluo Formula-containing serum exerts a protective effect against oxygen-glucose deprivation/reoxygenation(OGD/R)-induced injury in primary cortical neurons,and its mechanism may be associated with inhibiting the activation of the JNK signaling pathway and regulating the expression of apoptosis-related proteins and genes.