Effects Of Zhishu Pill Decoction-containing Serum On PI3K And Akt In Rat Colonic Interstitial Cells Of Caja

Objective: To observe the effect of medicated serum of Qizhu Pills on the proliferation of interstitial cells of Cajal in rat colon,and to explore its mechanism through the influence of PI3 K and Akt expression.Methods:Rat colonic Cajal interstitial cells were cultured in vitro,and 20serum-containing serum and blank serum were prepared from 20 SPF-class SD rats.The normal group,model group,blank serum group and Qizhu pills decoction group were established.CCK8 method was used to detect LY294002 intervention.The inhibition rate of cell proliferation,screening half of the inhibitory concentration,the same method to detect the optimal experimental concentration and time of the serum and blank serum of Qizhu pills,and the third generation of colonic interstitial cells according to the CCK8 test results,paved into 4 A dish.The normal group was cultured in serum-free complete medium for 24 hours.The model group,blank serum group and Zhizhuwan decoction group were treated with 25umol/L inhibitor LY294002 for 24 hours.The blank serum group and Qizhu pills decoction group were used.The medium containing the inhibitor LY294002 was aspirated,and the concentration of 10% blank serum and 5% drug-containing serum were added respectively for treatment for 24 hours.The morphology of colonic Cajal cells in each group was observed by microscope;RT-PCR,Western blot,immunization Fluorescence detection analysis of the effects of drugs on PI3 K,Akt protein and gene expression.Results:(1)CCK8 method showed that LY294002 showed a concentrationdependent inhibition of proliferation of rat colonic Cajal interstitial cells.The IC50 value of half inhibitory concentration was calculated according to SPSS software,and 25umol/L was the best experimental inhibitor concentration for24 h.5% phlegm and blood stasis decoction containing serum and 10% blank serum were the best experimental serum concentrations,and intervention for24 h was the best experimental time.(2)The growth of Cajal interstitial cells in rat colon was observed by inverted microscope.The results showed that the number of cells in the normal group was large,star-shaped,multi-protrusion,adherent growth and full morphology;rats after adding PI3K/Akt inhibitor LY294002 The growth of interstitial cells of Cajal in the colon was inhibited,and the number of cells was reduced.The residual cell processes became shorter and dull,and a complete cell network could not be formed.After the intervention of blank serum,the cells increased slightly and the protrusions became slightly longer.After the intervention of the drug-containing serum,the number of cells increased significantly,the neurites became longer,the branches increased,and the morphology became full.(3)Immunofluorescence assay: Compared with the normal group,the expression of PI3 K and AKT in the model group was significantly decreased(P<0.01).Compared with the model group,the expressions of PI3 K and AKT were significantly increased in the blank serogroup and Qizhu pills decoction group(P).<0.01);Compared with the blank serum group,the PI3 K expression in the Qizhu pills decoction group was statistically significant(P<0.01)and the AKT expression was increased(P<0.05).(4)Western blot analysis: Compared with the normal group,the expression of PI3 K and AKT in the model group was significantly decreased(P<0.01).Compared with the model group,the expression of PI3 K and AKT in the Qizhuwan decoction group was significantly increased(P<0.01).There was no significant difference in the expression of PI3 K and AKT(P>0.05).Compared with the blank serum group,the expression of PI3 K and Akt in the Qizhuwan decoction group increased(P<0.05).(5)RT-PCR detection: Compared with the normal group,the expression of PI3 K and AKT in the model group was significantly decreased(P<0.01).Compared with the model group,the expressions of PI3 K and AKT were significantly increased in the blank serogroup and Qizhu pills decoction group(P<0.01).Compared with the blank serum group,the PI3 K expression in the Qizhu pills decoction group was statistically significant(P<0.01),and the AKT expression was increased(P<0.05).Conclusion:(1)PI3K/Akt inhibitor LY294002 can inhibit the proliferation of Cajal cells,indicating that this pathway plays an important role in the development of interstitial cells of Cajal.(2)The medicated serum of Zhizhu Pill can promote the proliferation of interstitial cells of Cajal in rat colon,which may be related to the upregulation of PI3 K and Akt expression levels.(3)The medicated serum of Zhizhu Pill can promote the proliferation of interstitial cells of Cajal in rat colon,which may be related to the upregulation of PI3 K and Akt expression levels.