| ObjectiveAlcoholic liver disease(ALD)is a worldwide common liver disease.However,there is insufficient research on the treatment of ALD.The molecular mechanism studies of ALD and screening for effective drugs for prevention and treatment keep to be a hot research topic.The M1 polarization of Kupffer cells(KCs)induced by intestinal lipopolysaccharide(LPS)plays key roles in the pathogenesis of ALD.Nuclear factor E2-related factor 2(Nrf2)is a nuclear transcription factor regulating a battery of enzymes responsible for the elimination of reactive oxygen species(ROS).Nrf2 has long been one of the star molecules in the research field of life science and medicine.After activation,Nrf2 can antagonize the oxidative stress damage caused by alcohol exposure by upregulating the activation of the antioxidant system.In addition,recent studies have shown that Nrf2 can regulate the transformation of macrophages from M1 type to M2 type through crosstalk with NF-κB.Therefore,Nrf2 agonists have the potential to antagonize ALD.Diallyl disulfide(DADS)is a liposoluble organosulfur compound in garlic.The previous researches of our group found that DADS could effectively activate the Nrf2 signaling pathway and effectively inhibit the release of pro-inflammatory cytokines induced by LPS in RAW264.7 cells.However,it remains unclear whether DADS could regulate the M1 polarization of KCs by regulating NF-κB/MAPK inflammation-related signal pathways?It also remains unknown whether DADS could regulate the M1 polarization of KCs by regulating the NF-κB/MAPK inflammation-related signal pathways,reduce inflammatory reaction and antagonize alcoholic liver disease?Furthermore,is there a correlation between DADS regulation of KCs polarization and activation of the Nrf2 antioxidant pathway?In the current study,we firstly established the M1 polarization model of RAW264.7 cells and determined whether DADS could affect the secretion of inflammatory cytokines in RAW264.7 cells and measured the protein expression of NF-κB and MAPK signaling pathways using western blotting.Then,Nrf2-/-RAW264.7 cells were established by Crispr-Cas9 technique to explorethe effect of Nrf2 on the protective effect of DADS.Finally,the protective effect of DADS on LPS-induced liver inflammatory damage in mice was tested.Methods1.The intervention of DADS on LPS-induced Ml polarization of RAW264.7 cellsCCK-8 method was used to determine the dose range of DADS in RAW264.7 cells.The cells in the logarithmic growth phase were divided into 6 groups:control group,DADS(100μM)group,LPS(1μg/mL)group,LPS+DADS(25μM)low-dose group,LPS+DADS(50μM)medium-dose group and LPS+DADS(100μM)high-dose group.Each DADS treatment group was treated with different doses of DADS for 2h and the control group was given the same volume of complete medium.After that,LPS(1μg/mL)was added to continue cell culture for 24h.The levels of NO and TNF-a in the cell supernatant and the expression of key molecules in the NF-κB and MAPK signaling pathways in the cell was determined.2.The effect of knockout of Nrf2 on the protective effect of DADSCrispr-Cas9-mediated gene editing technique was used to estabolish Nrf2-knockout RAW264.7.Each of Knockout-control,Knockout Nrf2-1#and Knockout Nrf2-2#cells were divided into 6 groups:control group,DADS(100μM)group,LPS(lμg/mL)group,LPS+DADS(25.M)low-dose group,LPS+DADS(50μM)medium-dose group and LPS+DADS(100μM)high-dose group.DADS intervention group was treated with different doses of DADS for 4 h,and the control group was given the same volume of complete medium.After that,LPS(1μg/mL)was added to continue cell culture for 12 h.The cell culture medium was collected and used the Griess method to determine the NO content in the cell culture medium.3.Effects of DADS intervention onLPS-induced acute liver injuryForty male 8-week-old C57BL/6N mice were randomly divided into 4 groups(n=10)after one week of adaptive feeding including the control group,the LPS exposure group,the LPS+low-dose DADS groupand the LPS+high-dose DADS group.Mice in each DADS intervention group were given 15 mg/(kg.bw)(low-dose group)and 60 mg/(kg.bw)(high-dose group)DADS orally for 5 consecutive days,while mice in other groups were gavaged with same volume of corn oil.At 6 hours after the last dose of DADS or vehicle,LPS of 10 mg/(kg bw)were injected intraperitoneally to all mice except the control animals,which were injected intraperitoneally with the same volume of normal saline(NS).All mice were sacrificed 12 hours later.4.Determination of serum transaminase activityMouse blood were collected and centrifuged at 3500 r/m and 4℃ for 15 min using a low-temperature centrifuge to separate the serum.The activity of ALT and AST in serum were determined using commercial assay kits.5.Histopathological examination of mouse liverThe paraffin sections of mouse liver were made from formalin-fixed tissues,and H&E staining method was used to observe the liver damage.6.Measurementof NO,TNF-α,IL-1β and IL-6 levelsThe Griess method was used to determine the NO content in the cell supernatant.ELISA kit was used to determine the content of TNF-α,IL-1β and IL-6 in cell culture fluid and serum.7.Determination of the number of macrophages and neutrophils in mouse liver by immunofluorescence methodFrozen sections of mouse liver tissue were made and immunofluorescence technique was used to determine the makers of mouse liver macrophages(CD68)and neutrophils(Ly6G).8.Detection of the mRNA level of related molecules by qPCRThe liver tissue RNA was extracted and reverse transcribed into cDNA and the expression level of Ml polarization-related mRNA in the liver was determined by qPCR using SYBGREEN method.9.Measurement of the protein expression of related molecules by western blottingThe total protein samples of cells and liver were prepared using RIPA buffer and Western Blot method was used to determine the expression of NF-κB/MAPK/NLRP3 related inflammatory pathway proteins in cells and liver.Results1.The effect of DADS intervention on the secretion of NO and inflammatory factors in LPS-induced RAW264.7 cellsCompared with the control group,the content of NO and TNF-α in the cell culture supernatant of the DADS(100μM)group did not change significantly(P>0.05),but the content of NO and TNF-α in the cell culture supernatant of the LPS group increased significantly(P<0.05).Compared with the LPS group,the levels of NO and TNF-α in the cell supernatant of the LPS+DADS(25μM)low-dose group,the LPS+DADS(50μM)medium-dose group and the LPS+DADS(100μM)high-dose group all decreased(P<0.05).2.The changes of M1 polarization-related molecular proteins in RAW264.7 cells after DADS interventionCompared with the control group,the LPS group NF-κB signaling pathway associated protein p-IKKα/β,p-IκBα,IκBα,p-NF-κB and NF-κB increased significantly in the LPS group(P<0.05).Interestingly,the protein expression levels of p-IKKα/β,p-IκBaα NF-κB and p-NF-κB in the LPS+three-dose DADS group were significantly lower than those in the LPS group(P<0.05).Compared with the control group,after LPS induction,the expression levels of MAPK signaling pathway related proteins ERK,JNK and p38 total protein did not change significantly,but the expressions of p-ERK,p-JNK and p-p38 all increased significantly(P<0.05).Compared with the LPS group,p-ERK and p-JNK proteins were significantly reduced after the intervention of different doses of LPS+DADS(P<0.05),but the p-p38 protein expression changes were not statistically significant(P>0.05).3.The effect of NRF2 knockout on the protective effect of DADSCompared with the control group,the NO content of Knockout-control,Knockout Nrf2-1#and Knockout Nrf2-2#cells were significantly increased after LPS administration(P<0.05).The NO centent of the DADS(100μM)group did not change significantly(P>0.05).Compared with LPS group,NO content in knockout-control cells of LPS+DADS(50μM)medium dose group and LPS+DADS(100μM)high dose group was significantly decreased(P<0.05)and NO significant decrease in NO content in Knockout Nrf2-1#and Knockout Nrf2-2#cells(P>0.05).4.The effect of DADS intervention on LPS-induced acute liver injury in miceCompared with mice in the control group,the liver coefficient of the mice in the LPS group was significantly increased(P<0.05)and the activities of AST and ALT in the serum were significantly increased(P<0.05).Compared with the LPS group,the serum ALT and AST activities of mice in the DADS low-dose and high-dose intervention groups were significantly reduced(P<0.05).The results of liver histopathological examination showed that the liver tissues of the mice in the LPS group all had different degrees of liver damage,showing inflammatory cell infiltration and cell nucleus constriction.Interestingly,DADS low dose and high dose intervention to reduce the degree of liver injury group than LPS group.5.The influence of DADS on the secretion of serum inflammatory factors in LPS-induced miceCompared with the control group,the serum levels of TNF-α,IL-1β and IL-6 in the LPS group increased significantly(P<0.05).Comparedwith the LPS group,the secretion of IL-1β in the low-dose DADS intervention group was significantly reduced(P<0.05)and the secretion of TNF-α,IL-1β and IL-6 in the high-dose DADS intervention group was significantly reduced(P<0.05).6.DADS intervention changes the content of inflammatory cells in the liverThe results of immunofluorescence showed that there was no significant difference in the expression of CD68 in liver slices of mice in each group(P>0.05).Compared with the control group,significant Ly6G expression was observed in the LPS group(P<0.05)and Ly6G expression was reduced after DADS intervention(P<0.05).7.M1 polarization-related signal pathway protein and mRNA expression in the liver of DADS intervention in LPS-induced miceThe results of qPCR showed that the mRNA expression of iNOS,MCP-1,TNF-αand IL-6 in the liver of mice was significantly increased after LPS induction(P<0.05).Compared with the LPS group,the expressions of MCP-1 and IL-6 were reduced after the intervention of low-dose DADS(P<0.05).Obviously,the high-dose DADS intervention significantly inhibited the increase in the expression of iNOS,MCP-1,TNF-α and IL-6 mRNA(P<0.05).Compared with the control group,the total protein expression levels of NF-κB signaling pathway including p-IKKα/β,IκBa,p-IκBa,NF-κB,p-NF-κB,iNOS and COX2 increased significantly after 12h induced by LPS(P<0.05).Compared with the LPS group,after low-dose DADS intervention,the proteins of IκBα,P-IκBa,inos and COX2 were reduced(P<0.05).Afterhigh-dose(60mg/kg)intervention,the p-IKKα/β,IκBα,p-IκBα,NF-κB,p-NF-κB,iNOS and COX2 total proteins were significantly reduced(P<0.05).Compared with the control group,the expressions of MAPK signaling pathway related proteins ERK,JNK and p38 and their phosphorylated proteins p-ERK,p-JNK and p-p38 were not statistically significant after LPS induction(P>0.05).In addition,compared with the LPS group,low-dose DADS intervention p38 and p-ERK total protein expression slightly increased(P<0.05)and high-dose DADS intervention p-p38 protein expression slightly decreased(P<0.05).Compared with the control group,the expression of NLRP3 signaling pathway related proteins NLRP3,Cleaved-caspasel and Cleaved-IL-1β protein in the LPS group were significantly increased.Compared with the LPS group,both the DADS low-dose intervention group and the DADS high-dose intervention group could significantly attenuate the increase of NLRP3 inflammasome-related protein(P<0.05).Conclusion1.DADS can inhibit LPS-induced activation of NF-κB and MAPK signaling pathways in RAW264.7 cells by activating Nrf2 and inhibiting the secretion of inflammatory factors.2.DADS has a protective effect on LPS-induced acute liver inflammatory injury in mice,which may be related to the inhibition of NF-κB and NLRP3 signal pathway. |
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