Exploring The Molecular Biological Mechanism Of Cholesterol Gallstone With Liver Depression And Qi Stagnation Based On SCF/C-kit Pathway

ObjectiveThis study adopted the method of "2% high fat diet+chronic unpredictable mild stress(CUMS)+solitary feeding" to establish an animal model of cholesterol gallstone(CS)syndrome of liver stagnation and qi stagnation,and intervened with Chaihu Shugan Powder to explore the molecular biological mechanism of CS syndrome of liver stagnation and qi stagnation from the perspective of abnormal gallbladder SCF/C-kit signaling pathway inhibiting gallbladder contractile function,Provide a new entry point for the basic research of CS liver stagnation and qi stagnation syndrome.Methods1.Use the 2% high fat feed induction method to replicate the CS animal model.485-week-old SPF grade ICR male mice were randomly divided into a blank group(12 mice)and a model group(36 mice)according to their body mass.The blank group was fed with regular feed,while the model group mice were fed with 2% high-fat feed to establish a CS mouse model.After 16 weeks of feeding,three mice were randomly selected from each group to observe their gallbladder stones and determine whether the CS model was successful.After the success of the CS model,the mice in the modeling group were randomly divided into CS group,CS liver depression group,and Chaihu Shugan powder group(11 mice each)according to the body mass random grouping and zoning method.2.Establish an animal model of liver stagnation and qi stagnation syndrome using the "CUMS+solitary cultivation" method,that is,establish a CS liver stagnation and qi stagnation syndrome mouse model after 21 days of "CUMS+solitary cultivation".During the modeling period of liver stagnation and qi stagnation syndrome,the blank group continued to be fed with regular feed,while the other three groups of mice continued to be fed with 2% high-fat feed.On this basis,the CS liver stagnation group and Chaihu Shugan San group were fed in a separate cage and underwent 21 days of CUMS intervention to establish the CS liver stagnation and qi stagnation syndrome model.CUMS mainly includes fasting and water deprivation for 12 hours,tail clamping for 10 minutes,wet bedding for 24 hours,tilted cage for 45 ° 24 hours,and removal of bedding for 2-5 hours.One stimulus is randomly selected every day,and the same intervention method cannot be used continuously for 2 days.Before and after establishing the model of liver stagnation and qi stagnation syndrome,it is necessary to observe and record the general situation of each group of mice and conduct behavioral experiments.During the modeling period of liver stagnation and qi stagnation syndrome,the Chaihu Shugan San group was treated with modified Chaihu Shugan San by gavage,while the other groups were treated with equal amounts of physiological saline by gavage.After all interventions are completed,the mice are taken and the necessary samples for the experiment are collected.ELISA method was used to detect serum CCK content and bile TC,TBA,PL content,and calculate cholesterol saturation index(CSI).3.HE staining method was used to observe the morphology of gallbladder tissue.4.Immunofluorescence assay was used to detect the number of ICC cells in the gallbladder.6.The m RNA and protein expressions of gallbladder SCF,C-kit,CCK-A,PLC were detected using q-PCR and Western blot methods,respectively.Results1.General situation: During the period of 1-16 weeks,the mice in each group had normal diet and drinking water,were active,liked to climb,had good spirits,and had normal bowel movements.On the last day of the 16 th week,mice were weighed.Compared with the blank group,the CS group,CS liver depression group,and Chaihu Shugan powder group fed with 2% high fat diet showed significant body weight growth(P<0.01),and there was no difference in body weight among the three groups(P>0.05).During the modeling period of liver depression and qi stagnation syndrome,the CS liver depression group and the Chaihu Shugan powder group mice showed abnormal behaviors such as reduced body weight,mental fatigue,delayed activity,liking to curl up in corners,and no obvious resistance when grasping.Compared with the CS liver depression group,the Chaihu Shugan powder group mice showed a lighter degree of abnormal behavior and were more sensitive to stimuli than the CS liver depression group mice.After the modeling of liver depression and qi stagnation syndrome,compared with the CS group,the body mass of the CS liver depression group decreased significantly(P<0.01).Compared with the CS liver depression group,the body mass of the Chaihu Shugan San group increased(P<0.05).2.Behavioral experiment results: Before establishing the model of liver stagnation and qi stagnation syndrome,there were no differences in the results of forced swimming experiment,open field experiment,and sugar preference experiment among each group of mice(P>0.05).After the establishment of the model of liver depression and qi stagnation syndrome,compared with the CS group,the CS liver depression group showed a significant increase in stationary time and mid grid retention time(P<0.01),a significant decrease in upright times(P<0.01),a significant reduction in total movement distance(P<0.01),and a significant decrease in sugar consumption rate(P<0.01).Compared with the CS liver depression group,the mice in the Chaihu Shugan San group showed a significant decrease in resting time and mid grid retention time(P<0.01),an increase in upright times(P<0.01),a significant prolongation of total movement distance(P<0.01),and a significant increase in sugar water consumption rate(P<0.01).3.Gallbladder stones: After the establishment of the model for liver stagnation and qi stagnation syndrome,compared with the blank group,both the CS group and CS liver stagnation group mice showed brownish and turbid bile,poor transparency,increased gallbladder volume,and abnormal changes such as sediment like precipitation in the gallbladder.Compared with the CS group mice,the gallbladder volume of the CS liver depression group significantly increased,the degree of bile turbidity deepened,and the amount of sediment like precipitation in the gallbladder increased,with a wider range.Compared to the CS liver depression group,the Chaihu Shugan San group improved the degree of bile turbidity and transparency,reduced sediment like precipitation,and restored gallbladder volume in mice.4.Serum CCK content: Compared with the blank group,the CS group and CS liver depression group showed a significant decrease in serum CCK content(P<0.01).Compared with the CS group,the serum CCK content in the CS liver depression group was significantly reduced(P<0.01).Compared to the CS liver depression group,the serum CCK content in the Chaihu Shugan San group significantly increased(P<0.01).5.Bile TC,TBA,PL content,and CSI level: Compared with the blank group,the CS group and CS liver depression group showed a significant increase in TC content(P<0.01),while TBA and PL decreased significantly(P<0.01).The CSI level in the bile of both groups of mice was greater than 1(P<0.01).Compared with the CS group,the TC content in the bile of the CS liver depression group significantly increased(P<0.01),while the TBA and PL content significantly decreased(P<0.01).The CSI level in the bile of the CS liver depression group was significantly higher than that of the CS group(P<0.01).Compared with the CS liver depression group,the TC content in the bile of the Chaihu Shugan San group decreased(P<0.05),while the TBA and PL content increased(P<0.01,P<0.05).The CSI level in the bile of the Chaihu Shugan San group was significantly lower than that of the CS liver depression group(P<0.01).6.HE staining of gallbladder tissue: In the blank group,the gallbladder structure is intact,the thickness of the gallbladder wall is normal,and there are many folds and branches.The gallbladder of both CS group and CS liver depression group mice showed pathological changes such as thickening of the gallbladder wall,shortening of folds,and reduction of branches.Among them,the pathological changes mentioned above worsened in the CS liver depression group,with significant thickening of the gallbladder wall,short and thick folds,fewer branches,and severe morphological damage.The pathological changes such as thickening of gallbladder wall,shortening of folds,and reduction of branches in the Chaihu Shugan San group mice were significantly improved compared to the CS liver stagnation group.7.Number of gallbladder ICC cells: Compared with the blank group,the expression of gallbladder C-kit positive protein in the CS group and CS liver depression group decreased,and the number of gallbladder ICC cells significantly decreased.Compared with the CS group,the expression of C-kit positive protein in the gallbladder of the CS liver depression group was significantly reduced,and the number of gallbladder ICC cells was significantly reduced.Compared with the CS liver depression group,the expression of C-kit positive protein in the gallbladder of the Chaihu Shugan San group was significantly increased,and the number of gallbladder ICC cells was significantly increased.8.m RNA expression levels of gallbladder SCF,C-kit,CCK-A,and PLC: Compared with the blank group,the m RNA expression levels of gallbladder SCF,C-kit,CCK-A,and PLC in the CS group and CS liver depression group significantly decreased(P<0.01).Compared with the CS group,the m RNA expression levels of SCF,C-kit,CCK-A,and PLC in the CS liver depression group were significantly reduced(P<0.01).Compared with the CS liver depression group,the Chaihu Shugan San group significantly increased the m RNA expression levels of SCF and PLC(P<0.01),while the m RNA expression levels of C-kit and CCK-A increased(P<0.05).9.Gallbladder SCF,C-kit,CCK-A,and PLC protein expression levels: Compared with the blank group,the CS group and CS liver depression group showed a significant decrease in gallbladder SCF,C-kit,CCK-A,and PLC protein expression levels(P<0.01).Compared with the CS group,the expression levels of SCF,C-kit,CCK-A,and PLC proteins in the CS liver depression group were significantly reduced(P<0.01).Compared with the CS liver depression group,the expression levels of SCF,CCK-A,and PLC proteins in the Chaihu Shugan San group were significantly increased(P<0.01),and the expression level of C-kit protein in gallbladder tissue was significantly increased(P<0.05).Conclusions1."The chronic stress simulated by" CUMS+solitary confinement "for 21 days can significantly aggravate the condition of gallstones in CS models.".2.The low level expression of SCF and C-kit factors in the gallbladder SCF/C-kit signaling pathway inhibits the growth of ICC cells,leading to abnormal expression of CCK-A and PLC in the gallbladder,which may be the molecular biological mechanism of liver depression and qi stagnation syndrome in CS.3.Chaihu Shugan San can improve the low-level expression of SCF and C-kit factors in the gallbladder,promote the growth and development of gallbladder ICC cells,increase the expression level of CCK-A receptor,activate PLC expression,promote gallbladder contraction,reduce the degree of bile stasis,and play a role in soothing the liver,promoting gallbladder drainage,and removing stones.