| Objective:Previous studies in our group found that Zhizhu Pill Decoction can promote the proliferation and differentiation of Cajal stromal cells in mice with spleen deficiency and constipation,but the specific mechanism of Zhizhu Pill Decoction regulating Cajal stromal cells is stillunclear.Thisstudyfocused onthe PLC-gammasignaling pathway,took Cajalinterstitialcellsas the research object,and intervened Cajal interstitial cells with Zhizhu pill Decoction containing serum.The expression of PLC-γ1 and PLC-γ2 were detected.To explore the mechanism of Zhizhu Pill Decoctioninregulatinginterstitialcellsof Cajal.Methods:Cajalinterstitialcellsofmousecolonwereculturedinvitroandtreatedwith U73122,a specific inhibitor of PLC-γ,to block the signal pathway of PLC-γ.The blocked Cajal interstitial cells were intervened with serum containing Zhizhu pill Decoction.The blank serum group was set up as the control group.The rat colon interstitial cells treated with different factors were divided into normal group and model group(normal cells + 30umol/l U7312).2)blank serum group(model group + 10% blank serum)and Zhizhu pill group Decoction(model group + 5% Zhizhu pill containing serum).To observe the effect of Zhizhu Pill Decoction on interstitial cells of Cajal and to detecttheexpressionof PLC-γ-1and PLC-γ-2signalproteinandm RNAbyimmunohistochemistry,Westernblotand RT-PCR.Results:(1)U73122,a specificinhibitorof PLC-γ,caninhibittheactivityofmousecolon Cajalcells,and the inhibition rate increases with the increase of U73122 concentration.The inhibition rate is dose-dependent with U73122.When the concentration reaches 30 umol/l,it can achieve half inhibition.(2)(1)After 24 hours,there was no significant difference between the blank serum group and the normal group(P > 0.05),and there was no significant difference between the different concentrations of the blank serum group(P > 0.05).(2)After 24 hours,compared with the normal group,the number of interstitial cells of Cajal in different concentration of Zhizhu pill Decoction containing serum group increased significantly(P < 0.01);(3)After 24 hours,the proliferation rate of serum cells in different concentration of Zhizhu pill Decoction containingmedicinegroup was thehighest,andthedifferencewas statistically significant(P< 0.05).(4)There was no significant difference in the proliferation rate of interstitial cells of Cajal between the 10% serum group and the 5%serum group(P> 0.05).(5)Thehighest proliferation rate of interstitial cells of Cajal was observed 24 hours after the treatment with the 5% serum group containing Zhizhu Pill Decoction(P<0.01).(3)Therewere a large number of expressions of PLC-γ 1 and PLC-γ 2 in interstitial cells of colonic Cajal in normal rats.Compared with the normal group,the positive expression of PLC-γ 1and PLC-γ2 inthemodelgroupdecreasedsignificantly(P<0.01);comparedwiththemodelgroup,the positive expression of PLC-γ 1 and PLC-γ 2 in the Zhizhu pill Decoction group and the blank serum group increased significantly(P< 0.05);compared with the blank serum group,the positive expression of PLC-γ 1 and PLC-γ 2 in the Zhizhu pill Decoction group increased significantly(P<0.05).(4)The expression of PLC-γ1 and PLC-gamma PLC-γ 2 protein in colonic interstitial cells of normal mice was abundant.Compared with the normal group,the expression of PLC-γ1 and PLC-γ2 in the model group was significantly decreased,and there was significant statistical difference between the two groups(P < 0.01);Compared with the model group,the expression of PLC-γ1and PLC-γ2intheblankserumgroupand Zhizhupill Decoctiongroupwashigherthanthat in the model group(P< 0.05),and the expression of PLC-γ1 and PLC-γ2 in Zhizhu pill Decoction group was significantly higher than that in the model group(P < 0.01);Compared with the blank serum group,the expression of PLC-γ 1 and PLC-γ 2 protein in Zhizhu pill Decoction group was higherthanthatin Zhizhupillgroup Decoction,withstatisticaldifference(P<0.05).(5)There were a large number of PLC-γ 1 and PLC-γ 2 gene expressions in the interstitial cells of the colon of normal mice.Compared with the normal group,the expression of the target genes PLC-γ 1 and PLC-γ 2 in the model group was significantly decreased(P<0.01);compared with the model group,the expression of the target genes PLC-γ 1 and PLC-γ 2 in the blank serum group,Zhizhu pill Decoction group and the blank serum were significantly increased(P<0.01),but still significantly decreased compared with the normal group(P<0.01);the expression of the target genes PLC-γ 1 and PLC-γ 2 in the Zhizhu pill Decoction group and the blank serum were significantly increased(P<0.01);.The expression of PLC-γ 1 and PLC-γ 2 genes in Zhizhu Pill Decoctiongroupwashigherthanthatin Zhizhu Pill Decoctiongroup(P<0.01).Conclusion:(1).PLC-γ signaling pathway plays an important role in the development of murine colonic interstitialcells Cajal.(2)The serum containing Zhizhu pill Decoction could promote the proliferation of Cajal stromalcellsinmicecolon.(3)Theserumcontaining Zhizhupill Decoctioncanincreasetheexpressionof PLC-gamma1 and PLC-gamma 2 protein and the expression of m RNA.The intervention of Zhizhu pill Decoction containing serum on Cajal interstitial cells may be related to the regulation of PLC-gammasignalingpathway.(4)Regulation of PLC-gamma signaling pathway may be an important mechanism of Zhizhu Pilltopromotegastrointestinalmotility. |
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