Molecular Mechanism Of Isoliquiritigenin In Ameliorating Experimental Mouse Chronic Pancreatitis

Chapter?: Protective effects of isoliquiritigenin on chronic pancreatitis model in mouse.Objective:To establish the chronic pancreatitis(CP)model in mouse and observe the effects of isoliquiritigenin(ILG)on the pancreatic histology and the expression of fibrosis and inflammatory factors.Method:Mice were divided into control group,model group and treatment group,randomly.The chronic pancreatitis model in mouse was established by repeated intraperitoneal injections of caerulein and the mice in treatment group were given ILG by gavage in the last 3 weeks.Body weight was measured once a week and pancreas weight/body weight was recorded at the end.HE staining of tissue sections was used to determine the severity of the model and Sirius Red staining was used to show the extent of collagen deposition in the interstitial tissue of the pancrease.The mRNA levels of FN,?-SMA,COL-1,CCL2,CD68,F4/80,TNF-? and IL-6 in the pancreatic tissue were detected by PCR.In addition,the changes of blood glucose(Glu),alanine aminotransferase(ALT),total cholesterol(T-CHO)and triglyceride(TG)were used to observe the effects of ILG on the liver function and the histology of other organs(liver,lung,kidney and heart)were observed by HE staining.Results:The mean weight of mice in the model group was 21.4±0.81 g,compared to 24.2±0.66 g in the control group(p<0.01).While,the mean weight of mice in the treatment group was 22.9±0.42 g,which was higher than that in the model group(p<0.01).As for the pancreatic weight/body weight,the model group,control group and treatment group was 4.87±0.55,1.73±0.29 and 3.37±0.53,respectively.The differences between the model group and the control group(p<0.01)or the treatment group and the model group(p<0.01)were still statistically significant.The HE staining of the pancreases showed that the pancreatic tissues in the model were loose,the lobular structure was damaged,the acinar cells were significantly reduced and the ‘tubular complex' formed.A large production of fibrosis and infiltration of inflammatory cells were found in the interstitial tissue of the pancrease.There are a large number of fresh red cords and reticular collagen deposition in the acinar lobule.All these described above were alleviated in the treatment group for less acinar atrophy,collagen deposition and inflammatory cell infiltration.Besides,there were no such differences in Glu,ALT,T-CHO and TG among these three groups including the histological changes of liver,lung,kidney and heart.Conclusion:(1)The body weight and pancreatic weight/body weight in mouse CP model were significantly improved by ILG treatment.(2)ILG exerted the anti-fibrotic and anti-inflammatory effects by alleviating the severity of pathological changes in pancreatic tissue of the CP model and reduced the expression of fibrosis-related or inflammation-related factors,such as ?-SMA,FN,COL-1,CCL2,CD68,F4/80,TNF-?,and IL-6.(3)The serum expression levels of ALT,Glu,T-CHO and TG were not affected by ILG treatment,and the histological levels of liver,lung,kidney and heart were not affected either.Chapter? : The molecular mechanism of isoliquiritigenin inhibiting the activation of pancreatic stellate cells.Objective:To investigate the effects and the underlying mechanisms of ILG on the activation of PSCs for providing a new strategy of pancreatic fibrosis treatment.Method:CCK8 assay was conducted to detect the cell viability of ILG for a appropriate concentration range.ILG with gradient concentrations were used to treat PSCs with or without TGF-? and the group with DMSO was served as the control.The expression FN,?-SMA,COL-1 and CTGF were detected by PCR,Western blot and immunofluorescence.Wound healing assay,Transwell assay,and flow cytometry were conducted for observing the effect of ILG on PSCs migration and apoptosis.RNA-Seq technology was performed to identify the potential target genes and signaling pathways,which were verified furtherly.Results:The CCK8 assay showed that the suitable concentration of ILG was below 20?M.The PCR,Western blot and cellular immunofluorescence showed that FN,?-SMA,COL-1 and CTGF were suppresed in a dose-dependent manner by the ILG treatment.Wound healing assay showed that the migration distance of PSCs decreased after treating with ILG.Compared with the control group,the relative scratch width was 5?M: 1.04±0.17(p<0.05),10?M: 2.31±0.18(p<0.01),and 20?M: 3.70±0.23(p<0.01).Transwell assay also showed that the number of cells passing through the compartment membrane decreased significantly(5?M:72±13%,p<0.05,10?M:41±6%,p<0.01,20?M:14±3%,p<0.01).Flow cytometry showed that ILG promoted the apoptosis of PSCs in a concentration-and time-dependent manner.By RNA-Seq technology,we found that the expressions of IL1?,CCL2,and PDGFB related to the fibrosis and inflammation were down-regulated as well as the up-regulation in expression of DUSP5.Subsequently,we confirmed that ILG could significantly inhibited the phosphorylation of ERK1/2 and JNK1/2,which caused the suppresion the c-Jun nuclear translocation.Furthermore,we confirmed that ILG also decreased the phosphorylation level of PDGFR in PSCs and increased the expression of DUSP5 and DUSP10.Conclusion:(1)LIG inhibited the activation and migration of PSCs,while induced apoptosis.(2)ILG suppressed the expression of IL-1?,CCL2,and PDGFB,while up-regulated the DUSP5,and MAPK signaling pathway was involved in.(3)ILG-mediated dephosphorylation of ERK1/2 and JNK1/2 induced the inhibition on the activation of PSCs,which may caused by the dephosphorylation of PDGFR and the increased expression of DUSP5 and DUSP10.Chapter ? : The mechanism of isoliquiritigenin regulating the polarization of macrophages.Objective:To investigate the effect of ILG on macrophage polarization and its role in the crosstalk between PSCs and macrophage.Method:The CCK8 assay was conducted to test the effects of ILG on the RAW264.7 cell viability to achieve the appropriate concentration ranges.Then,LPS and IL-4 were used to induce different polarization of RAW264.7,respectively.The PCR and Western blot were used to separate the specific polarization of RAW264.7 including the relevant molecular markers.Then,the short-term LPS stimulation was used to activate the classical NF-?B pathway in RAW264.7 for observing the effects of ILG.The inflammatory factors in PSCs treated with ILG ware examed by PCR.The supernatant of PSCs were used to stimulate RAW264.7 for achieving the polarization changes and the effects of ILG.Results:The CCK8 assay showed that the suitable concentration of ILG on RAW264.7 was below 20?M.Induced by LPS,RAW264.7 increased expression of CD86,IL1-?,TNF-?,iNOS and IL-6 as showed by the PCR and Western blot and ILG pretreatment inhibited this effect.Induced by IL-4,RAW264.7 increased expression of Arg-1,CD206,CD301 and TGF-?,while the ILG pretreatment had no significant effects on these.After short-term LPS stimulation,the protein level of I?B? was decreased and the phosphorylation level of p65 and I?B? were increased in RAW264.7,indicating that NF-?B pathway was activated.ILG pretreatment suppresed the protein levels of I?B? and increased the phosphorylation levels of p65 and I?B in a dose-dependent mannar.By treated with ILG,the mRNA expression levels of CCL2,CXCL1,CCL5,IL-1?,IL-6 and TNF-? were down-regulated in PSCs.With the supernatant of PSCs,the expression of IL-1? and CD86 were increased in RAW264.7,which was further inhibited by ILG pretreatment and the protein level of Arg-1 and TGF-? were not affected.Conclusion:(1)ILG inhibited LPS-induced M1-type polarization of macrophages and no such significant effects were detcted on IL-4-induced M2-type polarization.(2)The inhibition of the classical NF-?B pathway by ILG is involved in the regulation of macrophages M1-type polarization.(3)ILG exerted the anti-inflammatory effect by suppressing the expression of inflammatory cytokines in PSCs and inhibition in PSCs supernatant induced M1-type polarization of macrophages.