| ObjectiveDiarrhea-predominant irritable bowel syndrome,belonging to the category of TCM spleen-deficiency diarrhea,is a chronic bowel disease with a recurrent pain,bloating,diarrhea as the main symptoms.IBS-D is the result from many factors such as the social environment,psychological,intestinal,genetic.IBS-D is related to immune dysfunction.Recent research suggests that stem cell factor/tyrosine kinase receptor signaling is an important regulator of IBS-D immune system dysfunction.Our previous preliminary study found that Changji'an formula had certain effect on IBS-D,but the further mechanism should be explored.This research aims to further explore the impact of Changji'an formula on IBS-D immune function and the role of SCF/C-kit system through animal experiments and cell experiments mutual authenticationt.To explore the relationship between SCF/C-kit signal and disorder of immune system in IBS-D rats,as well as the regulatory effect of Changji'an formula by animal experiments.By establishing IBS-D animal model with liver-depression and spleen-deficiency,to explore how Changji'an formula improves visceral hypersensitivity as well as liver-depression and spleen-deficiency in IBS-D rats.Besides,the effect of Changji'an formula on the immune system of IBS-D rats were tested,and the effect of Changji'an formula on SCF/C-kit system of IBS-D rats were verified.In addition,to explore the correlation between SCF/C-kit signal and visceral hypersensitivity,immune system in IBS-D rats,and then the relationship among pathogenic factors and SCF/C-kit system of IBS-D were analyzed.To further explore the effect of SCF/C-kit signal on biological function on IEC-6 and how Changji'an formula improves it through cell experiments.To investigate how SCF/C-kit signal regulates intestinal barriers by means of activating or blocking SCF/C-kit signal and establishing IEC-6 injured cell models for mutual authentication.To verify the regulatory effect and related mechanisms of Changji'an formula on SCF/C-kit signal in injured cell models which induced by LPS,and reveal the mechanism of action how Changji'an formula protects intestinal epithelial barriers.MethodsThe relationship between SCF/C-kit signal and disorder of immune system in IBS-D rats,as well as the regulatory effect of Changji'an formula1.IBS-D rat model was established by using separation of breast milk,stimulation of acetic acid and constraint of four limbs.All model rats were randomly divided into the model control group,the Pinaverium Bromide group(0.018 g · kg-1),the high dose of Changji'an group(33.48 g·kg-1),the medium dose of Changji'an group(16.74 g·kg-1)and the low dose of Changji'an group(8.37 g·kg-1),six in each group.All medication lasted for 14 successive days.Normal saline was given to another six normal rats in the normal control group.After medication,weight,daily food intake,sugar water preference and independent activity behavior characteristics of the rats were determined.The visceral sensitivity was assessed by rectum expansion experiments and the pathological features were investigated by HE staining.2.Normal SD rats were taken to establish IBS-D model by method of three factors.The model rats were then randomly divided into the model control group,the Pinaverium Bromide group,the high dose of Changji'an group,the medium dose of Changji'an group and the low dose of Changji'an group,six in each group.All medication lasted for 14 successive days.Normal saline was given to another six normal rats in the normal control group.After medication,thymus coefficient and spleen coefficient were determined.Morphological changes of intestinal epithelial cells were observed by using electron microscope.In situ expression of ICAM-1 and Akt was detected by immune-histochemical method.The content of tryptase,TNF-a.IL-8 and IL-10 in serum was detected by ELISA.The mRNA expression of TNF-a,IL-1?,NF-?B and Claudinl in colon was detected by real-time fluorescent quantitative PCR.3.Newborn SD rats were taken to establish IBS-D model by method of three factors.The model rats were randomly divided into the model control group and the medicine treatment group,six in each group.Another six normal rats was usd as the control group.The medicine treatment group was given Chang ji'an by intragastric administration for two weeks.Normal saline was given to rats in the model control group and normal control group in the same way.After medication,the rats were put to death.The colon was taken to detect the protein expression of SCF and C-kit by using immune-histochemical method.The gene expressions of SCF in hypothalamus and C-kit in colon were detected by using Q-PCR.The protein expressions of SCF and C-kit in colon were detected by Western Blot.4.The data normality of each index was analyzed by K-S analysis with a single sample.Product moment correlation coefficlent(Pearson correlation coefficients)was usd for the data of bivariate normaldistribution.Coefficient of rank correlation(Spearman correlation coefficients)was usd for data of non-bivariate normal distribution.The role of SCF/C-kit signal in biological function of IEC-6 and effect of Changji'an formula1.The rat intestinal crypt epithelial cells IEC-6 was cultured by means of the following four ways:(1)After giving SCF exogenously,cell proliferation was observed by RTCA.Cell morphology changes was observed by microscope.Gene expression of Claudinl?NF-?B and Akt were detected by Q-PCR.(2)After giving STI-571,cell proliferation was observed by RTCA.Cell morphology changes was observed by microscope.The effect of STI-571 on the protein expression of NF-?B in IEC-6 was detected by IF.The effect of STI-571 on IEC-6 Akt,NF-?B,Claudinl and CLaudin2 were detected by Q-PCR.(3)Cell damage model was established by LPS.Cell proliferation was observed by RTCA.The effect of LPS on the protein expression of NF-?B and C-kit in IEC-6 was detected by IF.The mRNA expression of SCF,C-kit,NF-?B,Claudinl and Claudin2 in inflammation model was detected by Q-PCR.(4)Inflammation model was established by TNF alpha.The mRNA expression of SCF,C-kit,NF-?B,Claudinl and Claudin2 was detected by Q-PCR.2.IEC-6 cells damage model was established by LPS.Suitable drug concentration was selected by RTCA,and was divided into high,medium and low dose group for intervention.The expression of SCF,C-kit and NF-?B in cell was investigated by using immune-histochemical and immune-fluorescence.The gene expression of SCF,C-kit,NF-?B,Claudinl and Claudin2 was investigated by PCR.ResultsThe relationship between SCF/C-kit signal and disorder of immune system in IBS-D rats,as well as the regulatory effect of Changji'an formula1.Compared to the normal group,there was no obvious interstitial edema in the colon mucosa and weight loss in rats of model group(P<0.01).Visceral sensitivity score increased(P<0.01).Average daily food intake,sugar water percentage preference and autonomous activity were lower(P<0.05).Compared to the model group,the weight of rats in Pinaverium Bromide group,the high dose of Changji'an group,the medium dose of Changji'an group was obviously increased(P<0.01).The score was significantly lower(P<0.01).The low dose was invalid(P>0.05).Daily food intake,sugar water preference percentage and autonomic activities in Pinaverium Bromide group,the high dose of Changji'an group,the medium dose of Changji'an group and the low dose of Changji'an group were improved significantly(P<0.05).2.Compared to the normal group,thymus coefficient and spleen coefficient of rats in the model group were significantly increased(P<0.05).There was no obvious change in the overall state of epithelial cells,but organelles were abnormal.The content of IL-8,TNF-a and tryptase increased(P<0.05).The content of IL-10 decreased(P<0.01).There was no obvious difference in the in situ expression of ICAM-1 and Akt.The mRNA expression of TNF-a,IL-1? and NF-?B increased(P<0.05,<0.01,P<0.05),but Claudinl significantly decreased(P<0.05).Compared to the model group,the thymus coefficient of each dose group decreased significantly(P<0.01).The spleen coefficient showed a downward trend,but there was no statistical difference(P>0.05).Pinaverium Bromide,high and medium dose of Changji'an could improve mitochondria and rough endoplasmic reticulum.The content of IL-10 in each medication group increased,and the difference was statistically significant(P<0.05,P<0.01,P<0.01,P<0.01).TNF-a decreased in the Pinaverium Bromide,high and medium dose of Changji'an groups(P<0.05,P<0.01,P<0.05).Tryptase in the medium and low dose of Changji'an groups decreased significantly(P<0.05,P<0.01).The mRNA expression of TNF-? in each medication group decreased significantly(P<0.05).The mRNA expression of IL-1? in the Pinaverium Bromide,high and medium dose of Changji'an groups(P<0.O1,P<0.01,P<0.05).The low dose group showed a downward trend,but there was no statistical difference(P>0.05).The mRNA expression of Claudinl showed a rising trend,but there was no statistical difference(P>0.05).3.Compared to the normal group,the positive expression of SCF and C-kit decreased in model group,protein expression of SCF and C-kit significantly decreased(P<0.01),the mRNA expression of SCF and C-kit significantly decreased(P<0.01).Compared to the model group,the positive expression rate of SCF increased in Pinaverium Bromide,no significant difference in the protein expression of SCF and C-kit(P>0.05),the mRNA of SCF and C-kit of significantly increased(P<0.05).The positive expression of SCF and C-kit in the high dose of Changji'an group increased.The protein expression of SCF in the high and low dose of Changji'an groups decreased(P<0.01,P<0.05).The expression of C-kit in the medium dose of Changji'an group significantly decreased(P<0.01).The mRNA expression of SCF in colon of high dose of Changji'an group significantly increased(P<0.01).There was no significant difference in other groups(P>0.05).4.The expression of SCF was negatively related with visceral sensitivity(P<0.01)and the spleen coefficient(P<0.01).The expression of C-kit was negatively correlated with thymus coefficient and spleen coefficient(P<0.01).The expression of SCF was inversely related with TNF-?,IL-8(P<0.01,P<0.05),and was positively correlated with IL10(P<0.05),and negatively correlated with the expression of C-kit and TNF-?(P<0.05).There was no correlation between SCF?C-kit and the expression of NF-?B and Claudinl(P>0.05).The role of SCF/C-kit signal in biological function of IEC-6 and effect of Changji'an formula1.(1)Different concentrations of SCF and the different time of IEC-6 had no effect on the cellular biological function.(2)When different concentrations of STI-571 stimulated IEC-6 for 4 hours,the cellular morphology was damaged to some degrees with concentration dependence.Different concentrations(100,50,25,12.5,6,3,1.5 ?mol/L)of STI-571 inhibited the cell reproduction to some degrees,much more in 100?mol/L and 50?mol/L.After different concentrations of STI-571 intervened IEC-6,the fluorescence intensity of NF-?B decreased significantly,much more in 50?mol/L.When inhibited by 50?mol/L and 100?mol/L,NF-?B mRNA expression of IEC-6 increased significantly(P<0.05,P<0.01),Claudinl significantly decreased(P<0.05,P<0.01).When inhibited by each dose of STI-571,there was no statistical difference in the mRNA expression of Akt and CLaudin2(P>0.05).(3)Different concentrations(100?g/ml,50?g/ml,25?g/ml)of LPS had an adverse effect on cell proliferation,with concentration dependence.IEC-6 did not express SCF.When LPS induced,in 50?g/ml and 100?g/ml,the NF-?B expression of IEC-6 increased,and the expression of C-kit decreased with concentration dependence.Compared to the normal group,IEC-6 inflammation model induced by TNF-?,when stimulated for one hour,the mRNA expression of NF-?B significantly increased(P<0.01),but there was no statistical difference in the mRNA expression of SCF,C-kit,Claudinl and Claudin2(P>0.05).(4)Compared to the normal group,IEC-6 inflammation model induced by TNF-a,when stimulated for one hour,the mRNA expression of NF-?B significantly increased(P<0.01),but there was no statistical difference in the mRNA expression of SCF,C-kit,Claudinl and Claudin2(P>0.05).2.The high dose of Changji'an group could increase the protein expression of C-kit and NF-?B in the inflammatory cell model induced by LPS.The high dose group could increase the mRNA expression of SCF,C-kit and Claudinl in the inflammatory cell model(P<0.01,P<0.05 P<0.01)and decrease the mRNA expression of NF-Kb(P<0.01).The medium group could increase the mRNA expression of SCF and Claudinl(P<0.05)and decrease the mRNA expression of NF-?B and Claudin2(P<0.05).The low dose could decrease the mRNA expression of NF-?B and Claudin2(P<0.05 and P<0.01).Conclusion1.It's proved that the rat model which was established by the method of three factors fits the characteristics of IBS-D of liver-depression and spleen deficiency,and the method of liver-smoothing and spleen-strengthening could improve visceral sensitivity and the symptom of liver-depression and spleen deficiency.2.The change of structure of immune organs and immune cells in IBS-D rats and the imbalance of inflammatory markers and the anomalous change of cellular structural protein.The method of liver-smoothing and spleen-strengthening could improve the function of immune organs and immune cells in IBS-D rats and could regulate the expression of inflammatory factors as well as improve the expression of structural protein of epithelial cells,which may be one of the action pathway for treatment of IBS-D.3.Changji'an formula could enhance the gene and protein expression of SCF and C-kit in colon of IBS-D,which reveal that Changji'an formula could regulate the SCF/C-kit system.The change of SCF/C-kit system of IBS-D has a certain degree of correlation with visceral hypersensitivity and immune disorders.4.Changji'an formula could regulate the SCF/C-kit system of IEC-6 cell induced by LPS,and at the same time,it could decrease the expression of NF-?B and Claudin2 and increased the expression of Claudinl. |
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