| ObjectiveLiver is the main metabolic site and the primary target organ when alcohol enters human body.Long-term heavy drinking can lead to alcoholic liver disease(ALD).Alcoholic fatty liver disease is usually reversible at the early stage of ALD.Without control and intervention,it can further develop into alcoholic hepatitis,alcoholic liver fibrosis and alcoholic cirrhosis.Severe alcoholism can even induce extensive hepatocyte necrosis,which can develop into liver failure and hepatocellular carcinoma.ALD is a multifactorial disease involving multiple pathogenesis?and there are no effective drug treatments at present.At this stage,there have been many studies and hypotheses about the mechanism of ALD.Among them,the activation of Kupffer cells and the oxidative stress in liver caused by ethanol exposure have been proved to play an important role in the pathogenesis of ALD.Kupffer cells(KCs)are resident macrophages in the liver,which play an non-negligible role in the occurrence and development of various liver diseases.Ethanol exposure causes a large number of enterogenous endotoxins to enter the liver,resulting in M1 polarization of KCs in the liver,which can release a large number of reactive oxygen species(ROS)and pro-inflammatory cytokines such as Tumor necrosis factor(TNF-a),etc.,thus causing liver damage.In addition,when excessive ethanol is metabolized in the liver,it can also produce a lot of reactive oxygen species and reactive nitrogen species under the action of cytochrome P450 2E1(CYP2E1)and NADPH oxidative enzymes(NOX),resulting in oxidative stress injury of the liver.Nuclear factor-E2-related factor 2(Nrf2)is an important target for regulating oxidative stress in the body,and it can regulate the expression of genes related to detoxification and antioxidant defense in the liver.Nrf2 can induce endogenous antioxidant defense by promoting the expression of many phase II detoxifying enzymes and antioxidant enzymes,which is one of the most important ways to promote cell antioxidant defense.Protecting liver damage caused by various chemicals,including ethanol,by activating the Nrf2 signaling pathway has become a new strategy to prevent liver diseases.Garlic oil(GO)is a garlic product produced by steam distillation,which has many beneficial biological activities.Experiments have proved that garlic has a variety of pharmacological effects,including antioxidant,anti-inflammatory,anti-cancer,immune regulation,lowering blood pressure and controlling blood sugar,as well as a certain protective effect on liver injury caused by various exogenous chemicals.The study found that GO can effectively inhibit ethanol-induced oxidative stress.It contains five major organosulfur compounds(OSCs),which have also been found to activate the Nrf2/HO-1 pathway in HepG2 cells.Then,can the organosulfur compounds in GO alleviate the secretion of inflammatory factors and oxidative stress induced by M1 polarization in Kupffer cells?What are the main components that play a major role?Is the mechanism related to Nrf2 signaling pathway?These are all uncertain questions.Thus,in this study,RAW264.7 cells were stimulated by LPS to establish a model of M1 polarization of KCs.Enzyme-linked immunosorbent assay(ELISA)and reactive oxygen species(ROS)kit were used to screen the effective components of five major organosulfur compounds in GO that can effectively alleviate oxidative stress and inflammatory factor secretion induced by KCs activation.Meanwhile,Western Blotting and qRT-PCR were used to detect the content of Nrf2-Keapl signaling pathway-related proteins and mRNA,to observe whether it can cause changes in Nrf2-Keapl signaling pathway,and to explore its possible role in the prevention of ALD,so as to provide new ideas and strategies for the prevention and treatment of ALD.Methods1.Cell Counting Kit-8 was used to detect the cytotoxicity of five main organosulfur compounds of GO in RAW264.7 cells.Three doses of high,middle and low were selected as the dosage within the safe dose range.2.RAW264.7 cells were divided into control group,LPS group and different doses of OSCs intervention groups.The control group did not receive any treatment.The RAW264.7 cells in LPS group were activated by 1 ?g/mL LPS.The intervention groups were incubated with different concentrations of OSCs for 2 hours,and then co-cultured with 1 ?g/mL LPS for 24 hours.The levels of inflammatory cytokines including TNF-alpha,IL-lbeta and IL-10 in cell culture supernatant were detected by ELISA,and the concentration of NO in cell supernatant was detected by Griess Reagent.According to the above results,we screened OSCs that could effectively inhibit the secretion of inflammatory cytokines and nitric oxide in M1 polarization RAW264.7 cells induced by LPS.3.DADS was Selected as the main research object,and 100 ?M DADS was used as the dosage.Total protein and total RNA were extracted at 0 h,3 h,6 h,12 h,24 h and 48 h of DADS treatment respectively;Western Blotting and qRT-PCR were used to detect the protein and mRNA expression levels of Nrf2,keap-1,NQO-1,SOD,gamma-GCSc,HO-1,LC3 and p62 at different times.4.RAW264.7 cells were treated with 0 ?M,2.5 ?M,5 ?M,12.5 ?M,25 ?M,50?M and 100 ?M DADS respectively.Total protein and total RNA were extracted 24 hours later.Western Blotting and qRT-PCR were used to detect the protein and mRNA expression levels of Nrf2,keap-1,NQO-1,SOD,gamma-GCSc,HO-1,LC3 and p62 under the intervention of different doses of DADS.5.RAW264.7 cells were divided into control group,LPS group and LPS+(high/medium/low)DADS groups.The control group was not treated with any treatment.LPS group was induced with 1 ?g/mL LPS.The other three groups were incubated with 25 ?M,50 ?M and 100 ?M DADS respectively.After 2 hours,these three groups were induced with 1 ?g/mL LPS.Total protein and total RNA were extracted 24 hours later.The protein and mRNA expression levels of Nrf2,keap-1,NQO-1,SOD,gamma-GCSc,HO-1,LC3 and p62 were detected by Western Blotting and qRT-PCR.Cell slides were prepared and the changes of Nrf2 protein were detected by immunofluorescent staining.Cell suspensions were collected after incubation with fluorescent probe to detect the content of ROS in cells.6.RAW264.7 cells were divided into 5 control groups and 5 DADS treatment groups.The control group was treated without any treatment.The DADS group was treated with 100 ?M DADS for 24 hours.Then 50 ?M cycloheximide was added to the 10 groups and the total protein was extracted after 0 min,15 min,30 min,45 min and 60 min respectively.The effect of DADS intervention on the degradation rate of Nrf2 protein was detected by Western Blotting.Results1.Safe doses of five OSCs:When RAW264.7 cell viability was above 90%in each dose group(P>0.05),it was considered that the cell viability was not significantly affected.The safe dose range of DATS is 0?25 ?M;the safe dose range of DADS is 0?150 ?M;the safe dose range of DAS is 0?1600 ?M;the safe dose range of AMTS is 0?100 ?M;and the safe dose range of AMDS is 0?200 ?M.DATS/DADS/DAS/AMTS/AMDS had no obvious toxicity to cells at this concentration.2.The effects of five OSCs on inflammatory cytokines secreted by LPS-induced RAW264.7 cells:The levels of inflammatory cytokines in cell supernatant of LPS group were higher than those of control group(P<0.05);the concentrations of NO,TNF-a and IL-lbeta in cell supernatant of DATS/DADS+LPS group were significantly lower than those of LPS group(P<0.05);only IL-lbeta in cell supernatant of DAS+LPS group decreased(P<0.05);the contents of NO and TNF-? in cell supernatant of AMTS+LPS group decreased slightly(P<0.05);the content of NO in cell supernatant of AMDS+LPS group decreased(P<0.05).3.The effects of different time and dose of DADS intervention on the levels of Nrf2 signaling pathway-related proteins and mRNA in RAW264.7 cells:Compared with the control group,the protein expression levels of Nrf2 increased significantly at 3 h,6 h,12 h and 24 h after DADS treatment(P<0.05),and increased with the increase of DADS intervention dose(P<0.05);the protein expression levels of keap-1 decreased significantly at 3 h,6 h,12 h,24 h and 48 h after DADS treatment(P<0.05),and decreased with the increase of DADS intervention dose(P<0.05).The protein expression levels of NQO-1,HO-1 and gamma-GCSc increased significantly at 3 h,6 h,12 h,24 h and 48 h after DADS treatment(P<0.05),and increased with the increase of DADS intervention dose(P<0.05);compared with the control group,the protein expression levels of SOD showed no significant difference at any time of any dose DADS treatment(P>0.05).The results of qRT-PCR showed that compared with the control group,the mRNA levels of Nrf2 was significantly decreased at 3 h,6 h,12 h and 24 h after DADS treatment(P<0.05),and also significantly decreased after different doses of DADS treatment(P<0.05);the mRNA levels of keap-1 was significantly decreased at 3 h,6 h,12 h and 24 h after DADS treatment(P<0.05),and also significantly decreased after different doses of DADS treatment(P<0.05).The mRNA levels of NQO-1,HO-1,GCLm and GCLc were significantly increased at 3 h,6 h,12 h and 24 h after DADS treatment(P<0.05),and at different doses of DADS treatment(P<0.05).Compared with the control group,the mRNA levels of SOD showed no significant difference at any time of any dose DADS treatment(P<0.05).4.The effects of different doses of DADS intervention on the content of Nrf2 signaling pathway-related proteins and mRNA in RAW264.7 cells induced by LPS:The results of Western blotting and qRT-PCR showed that,compared with the control group,the protein expression levels of Nrf2 and keap-1 in the LPS group were significantly decreased(P<0.05),and the mRNA levels of the two proteins were also significantly decreased(P<0.05).Compared with the LPS group,the Nrf2 protein expression levels showed an upward trend in DADS(low/medium/high)+ LPS group(P<0.05),while the protein expression levels of keap-1 decreased compared with the LPS group(P<0.05).And compared with LPS group,there was no significant difference in mRNA levels of the two proteins in DADS(low/medium/high)+ LPS group(P>0.05).Compared with the control group,the protein expression levels of NQO-1 and gamma-GCSc in LPS group decreased significantly(P<0.05),but the protein expression levels of HO-1 in LPS group increased significantly(P<0.05),and the protein expression levels of SOD in LPS group had no significant difference(P>O.05);compared with the control group,the mRNA levels of NQO-1,HO-1,SOD and GCLm in LPS group increased significantly(P<0.05),but the mRNA levels of GCLc in LPS group was significantly lower than that in control group(P<0.05).Compared with LPS group,the protein expression levels of NQO-1,HO-1 and gamma-GCSc in DADS(low/medium/high)+LPS group increased with the increase of DADS intervention dose(P<0.05),but the protein expression levels of SOD was not significantly different from that in LPS group(P>0.05);compared with LPS group,the mRNA levels of HO-1,GCLc and GCLm in DADS(low/medium/high)+ LPS group increased(P<0.05),but the mRNA levels of NQO-1 and SOD decreased(P<0.05).5.The effect of DADS on ROS contents in RAW264.7 cells:Compared with control cells,the intracellular ROS level in LPS group increased significantly(P<0.05),but there was no significant difference between DADS group and control group(P>0.05);the intracellular ROS content in DADS intervention group decreased in a dose-dependent manner compared with LPS group(P<0.05).6.The effect of DADS on Nrf2 in RAW264.7 cells induced by LPS:Compared with the control group,the protein expression levels of Nrf2 increased in DADS 100 ?M group;Nrf2 protein nuclear translocation was more obvious in LPS group than in control group;Nrf2 protein nuclear translocation was more obvious in DADS(low/medium/high)+LPS group than in LPS group7.The effect of DADS on the protein degradation rate of Nrf2 in RAW264.7 cells:When cycloheximide was added to inhibit the synthesis of Nrf2 protein,the degradation rate of Nrf2 protein in 100 ?M DADS group was significantly slower than that in control group.At 15 min,30 min,45 min and 60 min,the remaining Nrf2 protein contents was 53.4%,40.7%,23.3%and 14.1%in 100 ?M DADS group,respectively.Nrf2 protein contents remained 48%and 9.9%in the control group at 15 min and 30 min,respectively.At 45 min and 60 min,no Nrf2 was detected.8.Effects of DADS on the contents of autophagy-related proteins LC3 and p62 in RAW264.7 cells:Compared with the control group,the protein expression levels of LC3-?decreased significantly after 3 h,6 h,12 h,24 h and 48 h of DADS treatment(P<0.05),and decreased with the increase of DADS intervention dose(P<0.05);The protein expression levels of p62 significantly increased after 3 h,6 h,12 h,24 h,and 48 h of DADS treatment(P<0.05),and increased after 24 h with 50 ?M,100 ?M DADS treatment(P<0.05).The expression of LC3-? after LPS stimulation was not significantly different from the control group(P>0.05).And the protein levels of LC3-? had no statistically difference between LPS group and DADS(low/medium/high)+ LPS group(P>0.05).There was a significant increase in the protein levels of p62 after LPS stimulated compared with the control group(P<0.05),and DADS intervention following LPS stimulation significantly reduced the protein levels of p62 in a dose-dependent manner(P<0.05).Conclusion1.Among the five main OSCs contained in garlic oil,diallyl disulfide(DADS)has the strongest inhibitory effect on NO and inflammatory cytokines in the supernatant of RAW264.7 cells induced by LPS.2.DADS has protective effects on inflammation and oxidative stress induced by LPS-induced M1 polarization in RAW264.7 cells.The mechanism may be related to inhibiting the expression of Keap-1,decreasing the degradation rate of Nrf2,promoting Nrf2 into nucleus,and enhancing the expression of HO-1,NQO-1 and gamma-GCSc in RAW264.7 cells. |
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