Based On The Endoplasmic Reticulum Stress PERK/CHOP Pathway,the Mechanism Of Zhishi Regulation Of Rat Gastric Cajal Interstitial Cell Apoptosis Was Studied

Objective:To observe the effect of medicated serum on the endoplasmic reticulum stress-cell apoptosis induced by Tunicamycin(TM)in rat interstitial cells of Cajal(ICC)and to explore its mechanism.To further reveal the mechanism of action of Zhishi promoting gastrointestinal motility.Methods:Muscle layer was isolated from rat gastric antrum tissue,and ICC cells were extracted by enzyme digestion.After primary cultured gastric ICC cells were identified by fluorescence,ICC cells and drug-containing serum and related drugs were selected during logarithmic growth period.Co-culture.(1)Different concentrations of TM(0.1?g/mL,1?g/mL,10?g/mL,25?g/mL,50?g/mL,100?g/mL)and different durations(12 h,24 h,48 h,72 h)In the primary cultured ICC cells,a blank control group was set up,and then the effect of different concentrations of TM on the activity of ICC cells was detected by CCK-8 method;Hoechst 33342 staining and Annexin V-FITC/PI double staining flow method were used to detect the ICC of each group.Apoptosis;ultrastructural changes of cells after intervention by TEM;TM-induced expression of Bax,Bcl-2,CHOP and PERK mRNA in ICC cells induced by TM.(2)Preparation of medicated serum containing medicinal herbs,high-performance liquid phase method to identify its active ingredients and content;according to the optimum concentration of TM and the optimal time of action obtained in experiment(1),set blank control group,model group,Salubrinal group(endoplasmic reticulum stress inhibitor group),5%,10%,20%Zhishi containing drug serum group;CCK-8 method was used to detect the effect of each group on ICC cell activity;Annexin V-FITC/PI double staining flow The apoptosis of each group of ICC was detected by radionuclide method.The ultrastructural changes of cells were observed by transmission electron microscopy.The expressions of Bax,Bcl-2,CHOP and PERK mRNA in ICC cells were detected by RT-PCR.Results:(1)Compared with the control group,TM significantly reduced the cell activity of ICC(P<0.05).After TM intervention,ICC cells showed apoptosis,showing nuclear agglutination and pyknosis,and the apoptotic cell nucleus showed dense and bright blue fluorescence;and the apoptosis rate increased gradually with the action time,and the ICC cells were treated after 72 hours of intervention.The apoptotic rate has reached 54.5%;the volume of ICC cells becomes smaller after transmission electron microscopy,chromatin condensation,edge collection,lipid droplets and endoplasmic reticulum vacuolization,showing apoptotic bodies.The expression of Bax mRNA was up-regulated with the increase of TM concentration and intervention time;the expression of Bcl-2 mRNA was significantly down-regulated at 10?g/mL and 25?g/mL,and increased to normal level at 50?g/mL,when the concentration was 100?g/mL.The expression was significantly up-regulated,significantly down-regulated after 24 h of intervention,and increased to normal level after 48 h,and significantly up-regulated after 72 h,and was significantly different from each group(P<0.05);CHOP mRNA expression increased with TM concentration The up-regulation was gradually up-regulated with the prolongation of intervention time,and reached the highest at 48 h,while the expression was significantly down-regulated at 72 h(P<0.05);PERK mRNA expression was up-regulated with increasing TM intervention concentration.It was significantly up-regulated after 24 h of intervention time,and gradually decreased after 48 h and 72 h(P<0.05).(2)Synephrine,naringin and hesperidin were detected in the simmering decoction,and only synephrine was detected in the drug-containing serum.Compared with the control group,the cell viability of ICC in the model group was significantly decreased(P<0.05).Under the transmission electron microscope,the chromatin condensation and edge collection of ICC cells showed that apoptotic bodies were formed,and the expression of Bax and CHOP mRNA was significantly up-regulated(P<0.05),and there was no significant difference in Bcl-2 and PERK mRNA expression(P>0.05).After drug intervention,the cell activity of ICC was significantly increased(P<0.05),intracellular organelle damage was alleviated,no obvious apoptotic bodies were observed,apoptotic cells were decreased,and Bax and CHOP mRNA expression were significantly down-regulated,while Bcl-2,PERK mRNA expression was significantly up-regulated(P<0.05).Conclusion:TM can induce apoptosis of ICC cells,which may be related to endoplasmic reticulum stress;and Zhishi can inhibit TM-induced endoplasmic reticulum stress-apoptosis and enhance cell activity.The mechanism may stimulate the PERK/CHOP pathway with sputum,and inhibit the excessive endoplasmic reticulum stress and reduce apoptosis by inhibiting the expression of the proapoptotic gene Bax and increasing the expression of the anti-apoptotic gene Bcl-2.This may be one of the mechanisms of action to promote gastric motility.