Study On The Mechanism Of Paeoniflorin, The Active Ingredient Of Tiaogeng Decoction, In Regulating JNK Mitochondrial Signaling Pathway To Mediate Apoptosis Of Primary Hypothalamic Neurons

Objective To explore the effect of Paeoniflorin(PF),an active ingredient of "Tiao-Geng-Tang",on damaged primary hypothalamic neuronal cells,and explore the mechanism of PF to inhibit apoptosis of hypothalamic neuronal cells.Method(1)Culture of rat hypothalamic neurons in vitro and neuronal identification in neonatal female SD rats: hypothalamic single cell suspension was prepared by trypsinization,morphological characteristics were observed by inverted phase contrast microscopy.After being labeled with glial fibrillary acidic protein(GFAP)and ?-tubulin III,the neurons were identified by immunocytochemical staining.(2)Screening of tributyltin chloride(TBTC)intervention concentration and intervention time by CCK-8 and flow cytometry: the neurons of hypothalamus cultured for 7 days were treated with different concentrations of TBTC(100~300?g/L)for 24 h and 48 h,CCK-8 method was used to detect cell viability,flow cytometry was used to detect apoptosis,and the modeling time and concentration of TBTC-induced hypothalamic neuronal apoptosis model were determined.(3)PF improved neuronal cell damage in hypothalamus: CCK-8 method was used to screen the optimal concentration and time of paeoniflorin.17?-estradiol was selected as positive control,then hypothalamic neuron cells were divided into 6groups: control group,TBTC group,17?-Estradiol + TBTC group,PF low dose+ TBTC group,PF medium dose + TBTC group,PF high dose + TBTC group were intervened respectively,through CCK-8 method,lactate dehydrogenase method,Hoechst 33258 immune cell staining method,flow cytometry,JC-1mitochondrial membrane potential to detect the effect of PF pretreatment on neuronal apoptosis and calculate the percentage of apoptosis.(4)Mechanism of PF on neuroprotection of injured primary hypothalamic neuronal cells: RT-PCR was used to detect the expression of anti-apoptotic gene Bcl-2 and proapoptotic gene Bax.Western blot was used to detect the expression of MKK4,p-MKK4,JNK,p-JNK,Bcl-2,Bax and caspase-3.Result(1)The primary hypothalamic neuron cells of the newborn female rats cultured grew well with a purity of 92.23% in this study.(2)Hypothalamic neuron cells were dose-dependently inhibited by different concentrations of TBTC.Flow cytometry showed that the apoptotic rate of hypothalamic neuron cells treated with 150?g/L TBTC for 24 hours was(42.6±6.6)%(P <0.01),the most suitable model for simulating hypothalamic neuronal apoptosis.(3)After pretreatment with PF(25,50,100 ?M),TBTC intervention showed that the cell viability of the 17?-estradiol group and PF(25,50,100 ?M)groups was significantly increased at 24 h and 48 h,respectively,17?-estradiol group:(85±8.3)% and(75 ± 8.2)%(P < 0.01),PF 25 ?M group:(71 ± 13.9)% and(62 ±13.7)%(P < 0.05),PF 50 ?M group:(79 ± 10.0)% and(69±10.2)%(P<0.01),PF100?M group:(88±10.2)% and(78±10.1)%(P<0.01),and the lactate dehydrogenase activity in each group of PF pretreatment was significantly reduced(P <0.01);Hoechst 33258 immunocyte staining showed that the pyknosis morphology and granulosa cells were significantly decreased in the 17?-estradiol group and PF group,and the concentration-dose correlation was observed(P<0.01).The results of flow cytometry showed apoptosis increased significantly to(42.9±2.5)% after TBTC treatment for 24 h,(P<0.01 vs control),and 50 ?M,100 ?M PF pretreatment and 17?-estradiol treatment significantly decreased the apoptosis rate to(33.8±2.7)%,(21.54±2.9)%,(23.6±3.2)%(P<0.01),JC-1mitochondrial membrane potential test results showed that 17?-estradiol and paeoniflorin 25 50 and 100?M groups pretreatment can suppress decrease in mitochondrial membrane potential(P <0.01).(4)After TBTC intervention,the expression of phosphorylated MKK4 and JNK proteins increased,and the expression level of p-JNK/JNK increased(P<0.05).After pretreatment with paeoniflorin,the expression of phosphorylated MKK4 and JNK protein decreased,the expression levels of p-MKK4/ MKK4 and p-JNK/JNK decreased(P<0.05),and the level of caspase-3 was down-regulated(P<0.01),which increased Bcl-2 m RNA levels decreased Bax m RNA levels and decreased Bcl-2/ Bax gene and protein ratio(P< 0.05).PF still reduced the expression levels of p-MKK4/ MKK4 and p-JNK/JNK after the treatment of JNK-specific agonists(P<0.05).Conclusion(1)TBTC concentration of 150?g/L and intervention for 24 h as a model for inducing apoptosis of hypothalamic neurons.(2)Pretreatment with PF(25,50,100 ?M)can ameliorate TBTC-induced apoptosis in hypothalamic neurons,suggesting that PF has an inhibitory effect on hypothalamic neuronal apoptosis.(3)PF may play a neuroprotective effect on TBTC-injured hypothalamic neuron cells by regulating MKK4-JNK-mediated mitochondrial signaling pathway,and inhibition of phosphorylation of MKK4 and JNK may reduce apoptosis of hypothalamic neuronal cells.