Changes Of RhoA/ROCK Signaling Pathway Molecular In Liver-stomach Disharmony Functional Dyspepsia Rats And Intervention Mechanism Of Simo Decoction

Objective: Observe the gastrointestinal motility status of FD rats and the intervention effects of Simo Decoction,detect the expression of Rho A,ROCK 1,MYPT 1 and CPI-17 in gastric tissues,and explore whether Simo Decoction could improve gastrointestinal motility and promote gastric emptying in FD rats by upregulating Rho A/ROCK signaling pathway;Observe the contractile capacity of ROCK1 low-expressing gastric smooth muscle cells(GSMCs)and the intervention effects of Simo Decoction,detect the expression of Rho A,ROCK 1,MYPT 1,p-MYPT1 and CPI-17 after treated with Simo Decoction.To investigate whether the contractile function of GSMCs is associated with Rho A/ROCK signaling pathway.The aim is provide experimental basis for exploring the mechanism of FD.Methods:1.Animal experiments: 2-3 months old SPF male SD rats were randomly divided into control group,model group,high dose group of Simo Decoction,medium dose group of Simo Decoction,low dose group of Simo Decoction and domperidone group.The Liver-stomach disharmony FD rats was replicated by tail clamping stimulation.After model making,the Simo Decoction group was fed 5.6 g/kg/d、2.8 g/kg/d、1.4g/kg/d intragastric administration,the domperidone group was fed 3 mg/kg/d domperidone intragastric administration,and the model group rats was fed equal amount of distilled water continuously for 14 d.Model making for 7 d and drugs intervention for 14 d.Observe the general living condition and measure the body weight.After the administration intervention,determine the gastric residue rate,intestinal propulsion rate and sucrose preference rate.The gastric histopathological changes were observed by hematoxylin-eosin(H&E)staining.The m RNA expression levels of Rho A,ROCK 1,MYPT 1 and CPI-17 were measured by Real-time PCR in gastric tissues.The protein expression levels of Rho A,ROCK 1,MYPT 1,and CPI-17 were determined by immunohistochemistry;The protein expression levels of Rho A and ROCK1 in gastric tissues were determined by Western blotting.2.Cell experiments: Rat primary GSMCs were isolated and cultured in vitro and identified by Immunohistochemical experiments.Construction of ROCK low-expressing GSMCs using RNA Interference Technology.ROCK low-expressing GSMCs were treated with 0.075 μg/m L,0.0375 μg/m L,0.018 μg/m L Simo Decoction culture medium.Normal GSMCs were used as a control to detect the cell contraction effect under a microscope.The m RNA expression levels of Rho A,ROCK 1,MYPT 1 and CPI-17 were determined by Real-time PCR;The protein expression levels of Rho A,ROCK1,MYPT 1 and p-MYPT1 in gastric tissues were determined by Western blotting.Among them,the ratio of p-MYPT 1 to MYPT 1 protein content can indicate the activity of ROCK 1.Results:1.Animal experiments: The identification results of the animal model showed that compared with the normal group,the rats in the model group had rough fur,high alertness,nervous irritability,poor general survival condition.The body weight,sucrose preference rate and the intestine propulsion all decreased significantly,and the gastric residual rate increased significantly(P<0.01).The gastric histopathology showed no obvious abnormalities.The m RNA expression levels of Rho A,ROCK 1,MYPT 1 and CPI-17 were significantly reduced in gastric tissues(P<0.01).The results of immunohistochemical experiments showed that the precipitation containing brown particles were mainly distributed in the rat gastric smooth muscle layer,the average optical density value of Rho A,ROCK 1,MYPT 1,and CPI-17 in the rat gastric smooth muscle layer was significantly reduced(P<0.01).The protein expression levels of Rho A and ROCK 1 in the model group were significantly reduced compared with those in the normal group(P<0.01).After treatment of Simo Decoction,the rats in the high,medium and low dose groups were improved.2.Cell experiments: GSMCs with low expression of ROCK1 were successfully constructed.The results of cell shrinkage experiment showed that the GSMCs length was significantly shorter in the ROCK1 low-expression group(P<0.01).The m RNA expression levels of Rho A,ROCK 1,MYPT 1,and CPI-17 were significantly reduced in the ROCK1 low-expression group(P<0.01).The protein expression levels of Rho A and ROCK1 was significantly reduced in the ROCK1 low-expression group.ROCK1activity(p-MYPT 1/MYPT 1)was significantly reduced(P<0.01).After treatment with different doses of Simo Decoction,the abnormal changes of GSMCs in the high,medium and low dose group were improved and showed dose dependence.Conclusions:1.FD gastrointestinal motility dysfunction and GSMCs contraction disorders may be closely related to the Rho A/ROCK signaling pathway,and it is speculated that the Rho A/ROCK signaling pathway may be related to the pathogenesis of FD.2.Simo Decoction can promote rat gastric emptying and regulate the contraction of rat GSMCs.The mechanism of action may be related to the Rho A/ROCK signaling pathway molecules Rho A,ROCK 1 and the related effector targets MYPT 1、p-MYPT1 and CPI-17.