The Study Of Chaihushugansan On Effect And Mechanism Of Autophagy Of Rat Gastric Interstitial Cells Of Cajal In Vitro

Objective:To study the effect and mechanism of Chaihushugansan on the autophagy of rat gastric interstitial cells of Cajal in vitro, for further investigation on the mechanism of Chaihushugansan in promoting gastrointestinal motility.Methods:The Chaihushugansan containing serum was prepared according to serum pharmacology on SD rats. ICCs were identified by immunefluorescence and then the ICCs which on logarithmic growth phase were divided into different groups to co-culture in vitro with different drugs and serum containing herbs. (1) The ICCs’inhibition in different concentrations of glutamate (high/medium/low concentrations respectively were 10 mmol/L,5 mmol/L and 2.5 mmol/L) at 24h was detected by CCK-8 method. Western blot was used to detect the expression of autophagic protein LC3 after ICCs were co-cultured with different concentrations of glutamate at different times (3 h,6 h, 24 h). The electron microscope was used to observe ICCs’ultrastructure and autophagic vacuoles and immunofluorescence technology was used to measure the fluorescence intensity of autophagic protein LC3 after co-cultured with medium concentration of glutamate. (2) Based on the experiment (1) finding out the optimum concentration of glutamate and the optimum time, then setting a blank group,10% Chaihushugansan group and 3-MA group (5 mmol/L). The ICCs’growth activity in different drugs and serum containing herbs at 24h was detected by CCK-8 method. The electron microscope was used to observe ICCs’ ultrastructure and autophagic vacuoles. Immunofluorescence technology was used to measure the fluorescence intensity of autophagic protein LC3.The western blot was used to detect the expression of the proteins belong to autophagic signaling pathway, such as LC3/PI3KC3/Beclin1 and Bcl2.Results:(1) Compared with the control group, various concentrations of glutamate can significantly inhibit the active of ICC (all P<0.01). Among glutamate groups, the inhibiting effect of medium and high concentration group was stronger than low concentration group’s (all P<0.05). The LC3II/I of medium and high concentrations at the time of 3h/6h/24h, was significantly increased when compared with control group and low concentration (all P<0.01), but the medium concentration group at 3h was the best to increased the ratio of LC3II/I (all P<0.01). The medium concentration of glutamate showed obvious autophagic vacuoles and cytoplasmic vacuoles in ICC, and the autophagic protein LC3 fluorescence intensity of each ICC was significantly enhanced (all P<0.01). (2) Compared with the control group, the activity of ICC was significantly inhibited in the glutamate group (P<0.01). Chaihushugansan group and 3-MA group can significantly improve the growth activity of ICC when compared with glutamate group (all P<0.01). Under the electron microscope, the control group can be seen abundant mitochondria, endoplasmic reticulum, consistent with the literature. Glutamate group showed obvious autophagic vacuoles and cytoplasmic vacuoles in ICC, but not in the Chaihushugansan group and 3-MA group. Compared with the control group, the autophagic protein LC3 fluorescence intensity of each ICC in the glutamate group was more significantly enhanced (P<0.01), and it was also significantly different in 3-MA group’s (P<0.05); compared with glutamate group, the LC3 fluorescence intensity in Chaishugansan group and 3-MA group was significantly decreased (P<0.01). Compared with control group, the expression of proteins LC3II/I, Beclinl and PI3KC3 was significantly higher in glutamate group (all P<0.01), but it was lower on Bcl2 (all P<0.01); in the group of Chaihushugansan and 3-MA, the expression of proteins LC3II/I, Beclinl was higher than control group (all P<0.05), so was the PI3KC3 in 3-MA group (P<0.05). Compared with the glutamate group, the expression on protein LC3Ⅱ/Ⅰ, Beclinl and PI3KC3 of Chaihushugansan/3-MA groups was lower (all P<0.01), but the expression of Bcl2 was higher(all P>0.05). Compared with the 3-MA group, the expression of PI3KC3 in Chaihushugansan group had significant difference (P<0.05).Conclusion:Chaihushugansan can inhibit the autophagy induced by,glutamate on rat gastric ICC in vitro, and can increase ICC’s growth activity, the mechanism may be related to the inhibition of non dependent mTOR autophagic signaling pathway.It may be one of the mechanisms that Chaihushugansan promoting gastrointestinal motility.