Mechanism Of Curcumin Regulating JAK2/STAT3 Pathway And Macrophage Function In Non-alcoholic Fatty Liver Disease

Background: Non-alcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by excessive lipid deposition in hepatocytes caused by alcohol and other definite liver damage factors.An imbalance between lipid droplet formation rates,lipid droplet mobilization rates,and lipoprotein or bile acid secretion during the process can lead to excess lipid accumulation.NAFLD includes different histopathological stages,ranging from clinically asymptomatic hepatic steatosis to non-alcoholic steatohepatitis(NASH),and even liver fibrosis,cirrhosis,and eventually hepatocellular carcinoma.Curcumin is the main active polyphenol component in turmeric,and has a good therapeutic effect on neuroinflammatory diseases,rheumatic diseases,infectious diseases,malignant tumors,atherosclerosis and myocardial infarction,and has become a research hotspot.In recent years,many studies have shown that curcumin has good application prospects in the treatment of liver diseases,such as by acting on different types of cytokines,regulating various signaling pathways,exerting anti-fibrosis effects,inducing lipid accumulation and oxidative stress,and play a key role in inhibiting the occurrence and development of liver cancer.However,the exact mechanism by which curcumin protects against NAFLD remains unclear.Studies have found that the Januskinase/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway plays an important role in the occurrence and development of various inflammatory diseases.In liver diseases,ischemia-reperfusion injury,ischemic postconditioning and the anti-hepatic injury effects of some drugs are related to the activation of JAK2/STAT3 signaling pathway.In addition,activation of the JAK2/STAT3 pathway is closely related to resistance to cellular oxidative stress and maintenance of mitochondrial function.Therefore,we put forward the hypothesis that curcumin may play an anti-nonalcoholic fatty liver effect by activating the JAK2/STAT3 signaling pathway. In addition,we also explored the effect of curcumin on the development of liver fibrosis.Objective: 1.To clarify whether curcumin has the effect of resisting endoplasmic reticulum oxidative stress and cell apoptosis in non-alcoholic fatty liver cells,and to further clarify whether curcumin exerts the protective effect against non-alcoholic fatty liver by regulating JAK2/STAT3 signaling pathway;2.To explore the effect of curcumin on the occurrence and development of liver fibrosis,to clarify whether curcumin can inhibit the abnormal activation of hepatic stellate cells during the occurrence and development of non-alcoholic fatty liver disease,and to preliminarily describe its inhibitory mechanism.Method:Part 1:1.In vivo experiment: 8-10-week-old C57BL/6J male mice(18-22g)were adaptively fed for 1 week and then randomly divided into control group(Control group),control+curcumin group(Control+Cur group),High-fat diet group(HFD group)and high-fat diet+curcumin group(HFD+Cur group),10 animals in each group,Control group and Control+Cur group were fed only conventional feed,and the other two groups were fed 60% high Caloric diet induced fatty liver.After 6 weeks of feeding,the Control+Cur group and the HFD+Cur group were given curcumin by gavage(150 mg/(kg·d))for 4weeks,and the other two groups were given an equal volume of 0.9% normal saline as a control.After the modeling was completed:(1)DHR-ROS was injected into the tail vein,and the mouse liver was photographed with a fluorescence microscope to observe the oxidative stress of the living liver.(2)Sacrifice the mice to collect specimens: collect peripheral blood,and measure ALT,AST,inflammatory cytokines(IL-1β,IL-6,TNF-αchemokines(CCL2,CCL3,and CCL5)by ELISA);H&E staining,oil red staining and Sirius red staining were used to determine TG,MPO,ROS in liver tissue by ELISA,and COL1A1 m RNA by PCR.Flow cytometry was used to quantify neutrophils and apoptotic cells.2.In vitro experiments:(1)Non-alcoholic fatty liver cell model was made by stimulating primary liver cells with oleic acid,and divided into four groups according to different concentrations of curcumin: oleic acid group(Oleic group);oleic acid+curcumin1 μM group(Oleic+Cur 1 μM group);Oleic acid+Curcumin 4 μM group(Oleic+Cur 4 μM group);Oleic acid+Curcumin 8 μM group(Oleic+Cur 8 μM group).ELISA method was used to detect the quantification of ALT,AST and cellular TG in cell culture supernatant,CCK8 method to detect cell viability,lipid fluorescence staining and lipid fluorescence intensity quantification,Western blotting method to detect the expressions of BCL2,BAX,P-JAK2 and p-STAT3 Quantitative analysis was carried out according to the expression gray value of protein bands.(2)The non-alcoholic fatty liver cell model was made by stimulating primary liver cells with oleic acid.According to the different treatment methods of transfection with JAK2 si RNA first and then adding curcumin to intervene,they were divided into four groups: oleic acid group(Oleic group),curcumin group+oleic acid group(Cur+Oleic group);JAK2 si RNA+oleic acid group(JAK2 si RNA+Oleic group);JAK2 si RNA+curcumin+oleic acid group(JAK2 si RNA+Cur+Oleic group).ELISA method was used to detect ALT,AST,IL-1β,IL-6,TNF-α in cell culture supernatant,CCK8 method to detect cell viability,PI staining and quantitative analysis of fluorescence images to evaluate the degree of cell apoptosis,Western blotting method to detect P-The protein expression levels of JAK2,p-STAT3,GRP78,and p-e IF2α were quantitatively analyzed according to the gray value of the protein bands.Part 2:1.In vivo experiment:(1)Male mice aged 8-10 weeks were randomly divided into Control group(Control group),high-fat diet group(HFD group)and high-fat diet+curcumin group(HFD+Cur group),with 20 mice in each group.The control group was only fed with conventional diet,and the other two groups were fed with high-calorie diet to induce fatty liver.After 6 weeks of feeding,HFD+Cur group was given curcumin by intragastric administration for 4 weeks,and the other 2 groups were given equal volume of 0.9% normal saline as control.After modeling,the recruitment of macrophages in each group was detected by flow cytometry,and the flow data were quantitatively analyzed.The polarization of macrophages was detected by flow cytometry in HFD group and HFD+Cur group respectively.Macrophages were separated from HFD mice and HFD+Cur mice by flow sorting,and the macrophages were collected.Total RNA was extracted by lysis,and the level of IL-10 m RNA was detected by PCR.(2)After the model of NAFLD mice was successfully established,curcumin was given to the stomach for 4weeks in the same way as before,and then divided into CLL group and PBS group.The former was given intravenous injection of CLL every other day,while the latter was compared with PBS.IL-10 m RNA was detected by PCR,and Sirius red was stained for quantitative analysis.(3)NAFLD model mice were randomly divided into Anti-IL-10 group and Isotype group.The former was given Anti-IL-10 tail vein injection,while the latter was given Isotype control.Sirius red staining was used to detect the degree of liver fibrosis in mice.2.In vitro experiment:(1)The hepatic stellate cells sorted by flow cytometry were identified by GFAP fluorescence and cultured in vitro.The experimental group was intervened by IL-10 and compared with PBS.After 12 hours,the apoptosis of hepatic stellate cells was detected by flow cytometry and Ed U staining,and the migration and invasion ability of hepatic stellate cells were detected by Transwell experiment.(2)The sorted hepatic stellate cells were cultured in vitro,the activation of hepatic stellate cells was stimulated by oleic acid,the experimental group was induced by IL-10,and the control group was detected by PBS and α-SMA fluorescent staining.(3)Macrophages and hepatic stellate cells were co-cultured;The expression of MMP series protein was detected by Westernblotting.Results:Part 1:1.In vivo experiment:(1)H&E staining,oil red staining and Sirius red staining of paraffin sections of mouse liver showed that the pathological structure of HFD group showed that bullous fat became the main feature,the lipid deposition was more obvious than that of the control group,and the degree of fibrosis was aggravated;While HFD+Cur group was less severe than HFD group.(2)The concentrations of ALT and AST in peripheral blood of mice were detected,and the average lipid area and fibrosis degree of oil red O staining were quantitatively analyzed.Compared with Control group,HFD group was significantly higher(P < 0.05),while HFD+Cur group was significantly lower(P <0.05).(3)PCR showed that Col1A1 m RNA expression in HFD group was significantly increased(P < 0.05),while HFD+Cur group was significantly lower than that in HFD group(P < 0.05).(4)Flow cytometry was used to detect neutrophil recruitment,and enzyme-labeled instrument was used to detect MPO expression level in liver tissue,and HFD was significantly increased(P < 0.05),while HFD+Cur group was significantly lower than that in HFD group(P < 0.05)(5)The apoptotic cells were detected by flow cytometry and quantitatively analyzed.The number of apoptotic cells in the liver tissue of HFD group increased significantly(P<0.05),while that of HFD+Cur group decreased significantly(P < 0.05).(6)The levels of inflammatory cytokines and cytokines were detected by ELISA.Compared with HFD group,the levels of inflammatory cytokines(IL-1β,IL-6,TNF-α)in HFD+Cur group decreased significantly(P < 0.05).The levels of chemokines(CCL2,CCL3,CCL5)in HFD+Cur group also decreased significantly(P<0.05).(7)Mice were injected with DHR-ROS through tail vein,and observed and photographed by fluorescence microscope.The results showed that the degree of oxidative stress in HFD group was obvious,while that in HFD+Cur group was less than that in HFD group.The quantitative analysis of the data and the detection of ROS concentration in liver tissue by ELISA are consistent with the results of in vivo imaging.Compared with HFD group,the level of oxidative stress in HFD+Cur group decreased significantly(P <0.05).2.In vitro experiment:(1)NAFLD model was made by stimulating primary liver cells with oleic acid,and curcumin with different concentrations was given.The results showed that with the increase of curcumin concentration,the viability of hepatocytes also increased significantly(P < 0.05).The levels of ALT,AST,hepatocyte triglyceride and lipid fluorescence intensity in the culture supernatant decreased significantly(P<0.05).Western blotting showed that with the increase of curcumin concentration,BAX protein level decreased significantly(P<0.05),while BCL2,P-JAK2 and p-STAT3 levels increased significantly(P < 0.05).(2)After blocking JAK2/STAT3 signal pathway with JAK2 si RNA,the cell viability,p-JAK2 and p-STAT3 water decreased significantly(P <0.05).However,the levels of ALT,AST,IL-1β,IL-6 and TNF-α in the supernatant were significantly increased(P<0.05).The addition of JAK2 si RNA blocker significantly increased the apoptosis rate(P<0.05)and the expression levels of GRP78 and p-e IF2αprotein(P<0.05).Part 2:1.In vivo experiment:(1)There was no significant difference in macrophage recruitment between HFD+CUR group and HFD group.However,the polarization characteristics of macrophages(M1/M2 type)are obviously different.In HFD+Cur group,M1 type decreased significantly(P<0.05),while M2 type increased significantly(P<0.05).(2)The level of IL-10 m RNA in HFD+CUR group was significantly higher than that in HFD group(P<0.05).(3)After CLL was used to remove macrophages in mice,the quantitative analysis of fibrosis in CLL group increased significantly(P<0.05),and the level of IL-10 m RNA decreased significantly(P<0.05).(4)NAFLD model mice,after Anti-IL-10 intervention,there was no significant difference in the degree of liver fibrosis between Anti-IL-10 group and Isotype group(P<0.05).2.In vitro experiment:(1)The apoptosis of hepatic stellate cells in mice was detected by flow cytometry.Compared with the control group,the apoptotic cells in IL-10 group increased significantly(P < 0.05).Ed U fluorescence staining showed that the proliferation of hepatic stellate cells in L-10 group decreased significantly(P<0.05).(2)Transwell test was used to detect the motility of hepatic stellate cells.Compared with the control group,the migration and invasion of hepatic stellate cells in IL-10 group were significantly weakened(P<0.05).(3)Hepatic stellate cells cultured in vitro were stimulated by oleic acid,and α-SMA fluorescence staining showed that the fluorescence intensity of IL-10 group was significantly lower than that of the control group(P<0.05).(4)Macrophage and hepatic stellate cells were co-cultured,and MMP series proteins were detected by WB.The expressions of MMP9,MMP12 and MMP13 in macrophage and hepatic stellate cells co-cultured group were significantly higher than those in isolated culture group(P<0.05).Conclusion:1.Curcumin can significantly reduce hepatic steatosis,apoptosis,neutrophil recruitment and fibrosis induced by HFD in vivo.2.Curcumin significantly increased the activity of primary hepatocytes treated with oleic acid by activating JAK2/STAT3 signaling pathway,inhibited endoplasmic reticulum stress and apoptosis,and reduced hepatocyte injury and release of inflammatory factors.3.Curcumin can change from M1 type to M2 type by regulating the high expression of macrophages,and express IL-10,thus inhibiting the abnormal activation of hepatic stellate cells,promoting the apoptosis of hepatic stellate cells and inhibiting the motility of hepatic stellate cells.