| [Objective]Banxia Xiexin Decoction,as a classical prescription,has a wide range of effects on digestive tract diseases in modern medicine.Cajal interstitial cells have been widely studied as a hot interstitial cell target in recent years and have been found to be actively involved in various digestive tract diseases.Our research group has previously verified the effect of Banxia Xiexin Decoction on interstitial cells of cajal and related factors in vivo animals.However,the cells and various signaling pathways involved in gastrointestinal motility are complex,and the molecular mechanism involved in interstitial cells of cajal is still unclear.In this experiment,starting from the effect of Banxia Xiexin Decoction on apoptosis-related proteins of interstitial cells of cajal,the possible mechanism of Banxia Xiexin Decoction in treating " hypochondrium fullness" by affecting apoptosis of interstitial cells of cajal is discussed.[Methods](1)preparing drug-containing serum of Banxia Xiexin decoction:feeding rats in groups,and respectively obtaining drug-containing serum and animal serum containing deionized water by gavage;(2)isolating and culturing gastric cajal cells of newborn BALB/C mice,immunofluorescence double-labeled c-kit protein and ANO1 protein,and identifying the cells;(3)After stably culturing the cells,they were randomly divided into three groups:blank group(containing fetal bovine serum),animal serum containing deionized water as normal control group,and Banxia Xiexin Decoction drug serum group.The effects of each group on proliferation and apoptosis rate of interstitial cells of cajal were detected by cck8 method and flow cytometry.(4)ELISA enzyme-linked immunosorbent assay was used to detect the expression of eNOS endothelial nitric oxide synthase in cell supernatants of each group.The content of NO in nitric oxide was detected by biochemical method.(5)The expression of SCF protein,c-kit protein,Bcl-2 protein,Bax protein,caspase3 protein,NF-?B protein and PI3K protein in each group were detected by western blot Western blot.(7)Real-time fluorescence quantitative method was used to detect the effect of Banxia Xiexin Decoction on the expression of SCFmRNA,c-kitmRNA,Bcl-2mRNA,Bax mRNA,caspase3 mRNA,NF-kBmRNAand PI3KmRNA in each group.[Results](1)cell identification:the positive expression of c-kit protein and ANO1 protein were identified by immunofluorescence microscope.(2)comparison of interstitial cell activity of cajal between different groups:compared with blank group and normal group,Banxia Xiexin decoction group has no obvious effect on proliferation of interstitial cell of Cajal(p>0.05);At 24 hours,the cell activity of Banxia Xiexin Decoction group reached its peak value.(3)apoptosis rate:compared with blank group(7.667± 1.699)and normal group(6.3±2.317),there was no significant difference in 5%low concentration group(5.983±1.665)(p>0.05).(4.733±1.397)in the drug-containing group was significantly lower than that in the10%medium concentration group(P<0.05).There was no significant difference between the drug-containing group and the 15%high concentration group(5.35±1.139)(P>0.05).(4)biochemical method(enzyme colorimetric)and ELISA method were used to detect the expression of NO and eNOS in the cell supernatant:The expression of NO in each group was compared with that in the blank group(45.88± 11.89)and that in the drug-containing group(25.81 ±6)(P=0.001<0.05),which was significantly different from that in the normal group(47.04±5.21)(p<0.05).There was no significant difference between the blank group and the normal group(P>0.05).Comparison of eNOS expression in each group:Compared with the blank group(56.49±23.09),the expression of eNOS(28.43±13.25)in the drug-containing group was decreased(P=0.02<0.05),and compared with the normal group(56.16±19),the expression of eNOS in the drug-containing group was decreased(P=0.02<0.05),and the difference was statistically significant.There was no significant difference between the normal group and the blank group(P>0.05).(5)Western blot was used to detect the expression of apoptosis-related proteins in interstitial cells of cajal:Compared with the blank group,there was no difference in protein expression between the normal group and the blank group(P>0.05).There was no significant difference in the expression of c-kit protein(P=0.28>0.05)in the drug-containing group,but there was no significant difference in the expression of NF-Kappa B(P = 0.074>0.05)in the drug-containing group,and there were significant differences in the expression of protein in other groups.Compared with the normal group,the expression of c-kit protein(P=0.212>0.05)in the drug-containing group had no statistical significance,while the NF-Kappa B(P?0.064>0.05)in the drug-containing group had no statistical significance,while the other groups had significant differences.(6)RT-PCR detection of apoptosis-related gene expression in interstitial cells of cajal:The blank group is set as the reference group and the CT value is set to 1.Compared with the normal group(1.711±0.382),the drug-containing group C-c-kit(2.304±0.378)has significant difference(P<0.05)with statistical significance.Bcl-2(1.416±0.204)was significantly different from that of the normal group(0.819±0.257)(P<0.05).SCF(1.253±0.372)in drug-containing group had no statistical significance compared with normal group(0.869±0.376)(P>0.05).Bax(1.108±0.397)in the drug-containing group was significantly different from that in the normal group(1.428±0.111)(P<0.05).Caspase 3(1.359 ±0.385)in the drug-containing group was significantly different from that in the normal group(1.921?0.243)(P<0.05),with statistical significance.Compared with normal group(1.626±0.292),PI3K(1.003±0.152)in drug-containing group has significant difference(P<0.05)with statistical significance.The NF-? Bp65(0.896±0.273)in the drug-containing group was significantly different from that in the normal group(1.419±0.339)(P<0.05).[Conclusion](1)Banxia Xiexin Decoction inhibits apoptosis of interstitial cells of cajal;24 hours,10%concentration of the whole prescription of Banxia Xiexin Decoction SD rat Banxia Xiexin Decoction containing drug serum culture is obviously beneficial to inhibit interstitial cells of caj al.(2)Banxia Xiexin Decoction can obviously inhibit the secretion of eNOS and NO by interstitial cells of caj al.(3)Banxia Xiexin Decoction can promote the expression of SCF and Bcl-2 protein in interstitial cells of cajal and reduce the expression of caspase3,Bax and PI3K in interstitial cells of cajal.Up-regulated expression of c-kit and Bcl-2 genes in interstitial cells of cajal,down-regulated expression of caspase3,NF-Kappa BP65,PI3K and Bax genes in interstitial cells of caj al.To sum up,Banxia Xiexin Decoction has certain effects on proteins and genes related to apoptosis of interstitial cells of cajal.Preliminary discussion may be related to the synergistic effect of SCF/c-kit signaling pathway and PI3K and NO signaling pathway.The specific synergistic mechanism between signaling pathways needs further in-depth study.The regulation of Banxia Xiexin Decoction on gastrointestinal motility may be closely related to the inhibition of apoptosis of interstitial cells of cajal. |
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