AP39 Ameliorates High Fat Diet-induced Liver Injury In Young Rats Via.alleviation Of Oxidative Stress And Mitochondrial Impairment

Background:Nonalcoholic fatty liver disease（NAFLD）in children and adolescents refers to chronic steatosis of liver in immature individuals（<18 years old）.In chronological order,from light to heavy.Nonalcoholic fatty liver（NAFL）was the lightest.This was followed by nonalcoholic steathepatitis（NASH）and the most serious Nash-related cirrhosis,some of which further developed into cirrhosis or even hepatocellular carcinoma.But with the deepening of understanding,medical and scientific research workers aware of NAFLD is a metabolic disease,in addition to the fatty degeneration of liver itself due to fat accumulation,liver cirrhosis,even with fat metabolic disorder caused damage of body organs,develop high cholesterol,insulin resistance,atherosclerosis and other more serious metabolism related cardiovascular disease.Due to the lack of understanding of the disease and the hidden clinical characteristics of the disease,more people ignore the harm brought by NAFLD.The pathogenesis of nonalcoholic fatty liver disease in children and adolescents has not been determined for the time being,and the popular viewpoints are"second strike theory"and"multiple strike theory".The first blow is certain,that is,the accumulation of fat in the liver,fatty acids into the mitochondria oxidative decomposition,and then synthesis of cholesterol,triglyceride.As we all know,NAFLD is simple fatty liver in the initial stage,and NASH in the second stage.Therefore,what exactly is involved in the process of adipose inflammation in the second stage,and whether HFD diet can be involved in the occurrence and development of NAFLD mechanism,the temporary mechanism is not clear.However,oxidative stress and lipid peroxidation involved in ROS are very hot topics in the study of"multiple strike theory".Therefore,if the pathogenesis can be found in the process of oxidative stress and lipid peroxidation,it will provide a clearer target for further intervention of NAFLD.It is possible to improve NAFLD by improving oxidative stress.For too long,hydrogen sulfide（H₂S）has played the bad role.In recent years,with the in-depth study of this gas,H₂S is a new endogenous gas transmitter.H₂S is involved in the regulation of many physiological functions in humans,including oxidative stress,glycolipid metabolism,cardiovascular system,differentiation,and circadian rhythm.Many studies have shown that H₂S is associated with the pathogenesis of liver diseases such as liver fibrosis,cirrhosis,hepatocellular carcinoma,hepatic ischemia/reperfusion injury,non-alcoholic fatty liver disease/non-alcoholic steatohepatitis,hepatotoxicity,acute liver failure and so on.H₂S alleviates oxidative stress through multiple signaling pathways,thereby reducing lipid accumulation in the liver.In recent years,H₂S has been gradually found to play a very clear role in protecting mitochondria.However,H₂S is a gas molecule,and its release concentration cannot be well controlled.If the concentration is too high,the toxic effect concentration may be achieved,while if the concentration is too low,the pharmacological effective concentration cannot be achieved.Moreover,the gas is volatile,difficult to locate in the body,and the concentration of the gas to the target organs of concern cannot be well controlled,which greatly limits the application of H₂S gas itself as a drug in basic research and clinical trials.Therefore,the application of H₂S is limited,so finding the right H₂S donor to study the mechanism between H₂S and NAFLD will not only help people to understand the role of H₂S in NAFLD,but also provide theoretical basis and guidance for clinical drug research.AP39（10OXo-10-（4-（3-thioxo-3H-1,2-Dithiol-5yl）phenoxy）decyl）is a mitochondrial H₂S-targeted donor.It is a derivative formed by combining 5-hydroxyphenyl-3H-1,2-dithiacyclopentene-3-thioneone compound（ADT-OH）with triphenylphosphine,a group with mitochondrial targeting,which can concentrate the donor in mitochondria and increase the concentration of H₂S in mitochondria.Studies have shown that AP39has protective effects on cardiac ischemia-reperfusion and Alzheimer’s disease.However,there is no application of AP39 in NAFLD currently.Objective:To establish a rat model of juvenile non-alcoholic fatty liver disease（NAFLD）,to investigate the effects of high fat diet on liver gross,liver function,serum lipid and other related serological indexes,and to determine H₂S level and oxidative stress markers.To investigate the effects of AP39 on mitochondrial function,HIF-1αand mt DNA in animal models of nonalcoholic fatty liver disease.Methods:Twenty-four healthy male 3-week-old SD rats were randomly divided into four groups:A:Control group,B:HFD group,C:HFD:HFD+L-AP39 group and D:HFD+H-AP39 group.The Control group was fed with standard diet,and the other three groups were fed with high-fat diet.The HFD group was injected with the same dose of normal saline as the AP39 group every day,and the HFD+L-AP39 group was injected with AP39 every day at the dose of 0.05mg/kg.In HFD+H-AP39 group,AP39was injected into tail vein every day at a dose of 0.1mg/kg.Body weight change and food intake were recorded once a week for 7 weeks.The weight changes of rats in the four groups were compared,and the differences of gross liver index,NAFLD activity score,liver HE staining and oil red O staining were recorded after modeling.ALT/AST,TC/TG,LDL-C/HDL-C,H₂S,MDA,SOD and GSH were determined respectively.DHE staining method was used to detect ROS levels,fluorescence microplate analyzer was used to detect mitochondrial membrane potential of SD rats,and the absorption value at 520nm was used to determine the degree of mitochondrial swelling.The copy number of mt DNA was detected by RT-PCR and HIF-1αprotein expression was detected by Western blot.Meanwhile,The m RNA level of HIF-1αwas detected by RT-PCR.Results:1.At week 7 of modeling,the body weight of SD rats in the four groups was291.5±54.1g in the Control group and 409.3±47.3g in the HFD group,respectively;HFD+L-AP39 group:354.5±30.1g;HFD+H-AP39 group:310.7±22.2g.The average body weight of HFD+H-AP39 group was almost close to that of Control group.HFD significantly reduced the food intake of rats from week 3 to week 7.By the end of week 7,the food intake of SD rats in HFD group was 20.8±3.3g,while that of SD rats in Control group was 26.2±4.1g.The food intake of AP39 treated SD rats showed an increasing trend except the fifth week,but was not significant.At week 7,the intake of HFD+L-AP39 group and HFD+H-AP39 group was 23.7±2.0g and 24.5±1.5g,respectively.2.The liver volume of HFD group increased significantly,NAFLD activity score was 9,and all of HFD group had the pathological features of typical NAFLD,such as hepatic steatosis,lobular inflammation and hepatocellular balloon degeneration.The liver of the rats treated with different doses of AP39 showed a yellow-red appearance,but its volume was larger than that of the normal liver,and the fat accumulation degree of the liver was significantly improved compared with that of the HFD group,and the yellow fat part was reduced.The mean liver index of normal diet group was 3.07±0.277.4.03+/-0.505 HFD group;HFD+3.49+/-0.265 L-AP39 group;HFD+3.25+/-0.155 H-AP39 group.3.The average liver function of normal eating control group was ALT:55.176±11.636U/L,AST:108.417±23.236,the liver enzyme was significantly increased in HFD group,ALT was176.263±43.82U/L,AST was 269.659±55.97U/L;Compared with HFD group,AST and ALT decreased significantly after AP39 intervention.4.TG\TC of normal eating control group were 0.821±0.208mmol/L and2.02±0.448mmol/L respectively.TG of HFD group:2.821±0.587mmol/L,TC:6.17±1.476mmol/L),TG/TC of THE two groups after AP39 intervention was significantly lower than that of HFD group.5.LDL-C in HFD group was significantly higher than that in control group（0.301±0.064mmol/L）.Compared with HFD group（1.007±0.222mmol/L）,there were significant differences between the two groups after AP39 intervention,and the difference was statistically significant.Compared with the control group（1.293±0.29mmol/L）,HDL-C in HFD group was significantly lower,and the difference was statistically significant.Compared with HFD（0.529±0.110mmol/L）group,AP39 showed significant difference between the two groups,and the difference was statistically significant.6.The H₂S level in the control group was 0.104±0.022umol/mg protein,while the H₂S level in the HFD group was significantly reduced（0.046±0.013umol/mg protein）,and the difference was statistically significant.Compared with HFD group,H₂S（0.076±0.018umol/mg protein）level of HFD+L-AP39 group increased after AP39intervention,and there was no statistical difference compared with HFD group.H₂S（0.091±0.019umol/mg protein）level in HFD+H-AP39 group was significantly increased,and the difference was statistically significant.7.MDA in the control group was 2.014±0.536nmol/Mg Prot,while that in the HFD group was significantly increased,with a value of 7.119±1.716nmol/mgprot,and the difference was statistically significant.Compared with HFD group,MDA significantly decreased after AP39 intervention,to 5.129±1.186nmol/mg Prot and4.046±1.047nmol/mgprot,respectively,and the difference was statistically significant.In the control group,GSH and SOD were 19.915±4.345μmol/gprot and69.868±14.286U/mg Prot,respectively.In the HFD group,GSH and SOD were significantly reduced to 6.772±1.927μmol/gprot and 27.096±6.013U/mg Prot,respectively.The difference was statistically significant.Compared with HFD group,GSH and SOD increased in the two groups after AP39 intervention,and the difference was statistically significant.8.DHE staining showed that cells in the normal control group only had low fluorescence intensity,while cells in the HFD group had strong red fluorescence.After L-AP39 intervention,ROS cell fluorescence intensity was lower than that in the HFD group,while ROS cell fluorescence intensity was lower in the HFD+H-AP39 group than that in the HFD group,and the number of red fluorescence cells was significantly reduced.9.Mitochondrial swelling can be measured by absorption at 520nm.The control group had mitochondria at 520nm absorbance value is 0.377±0.078.The absorbance value at520nm in HFD group was significantly decreased（0.125±0.032）,and positive Compared with the control group,the difference was statistically significant.The absorbance value of 520nm in HFD-L-AP39 group was 0.228±0.053.The absorbance value of 520nm in HFD-H-AP39 group was 0.298±0.070.The difference was statistically significant.10.MMP of control group was 2.479±0.652Δψm,MMP of HFD group was significantly lower,0.509±0.112Δψm,the difference was statistically significant;Compared with HFD group,there were significant differences between AP39 and HFD group,which were 1.279±0.333Δψm and 1.701±0.436Δψm,respectively,and MMP was significantly increased.11.Compared with the control group（1.0±0.04）,the m RNA level of HIF-1αin HFD group（4.0±0.16）was significantly increased,and the difference was statistically significant.Compared with HFD group,the m RNA levels of HIF-1αin AP39 group and HFD group decreased significantly（3.11±0.32 and 1.86±0.13,respectively）.12.Western blot was used to detect HIF-1αprotein expression.Compared with the control group,HIF-1αprotein expression level in HFD group was significantly increased（5.46）,and the difference was statistically significant.Compared with HFD group,hi F-1αprotein levels in AP39 group and HFD group decreased significantly（protein expression levels were 3.44 and 1.88,respectively）,and the difference was statistically significant.Conclusion:1.AP39 reduced HFD-induced weight gain in SD rats in a dose-dependent manner,and the weight gain of young rats in the high-dose AP39 group was slower than that in the low-dose AP39 group.AP39 could control the weight gain of SD rats fed with HFD.AP39 may have a potential role in childhood obesity.The liver index was reduced by AP39,which has a protective effect against hepatocyte injury induced by a high-fat diet in vivo.After AP39 treatment,the liver showed obvious steatosis,and the area of lipid infiltration and lipid vacuole in the liver increased,and the volume of liver cells increased.2.AP39 can improve liver function and reduce blood lipid,cholesterol and LDL-C in NAFLD rats.It can effectively inhibit lipid accumulation in HFD-fed young rats.On the contrary,HDL-C results were consistent with the changes in blood lipids of NAFLD patients,indicating that the animal model of NAFLD not only had pathological changes and lipid deposition,but also had changes in liver function and blood lipids,and was consistent with the serological changes of human NAFLD.3.AP39 can increase H₂S level in HFD+H-AP39 group and HFD+L-AP39 group.In addition,AP39 can reduce MDA level;The levels of GSH and SOD activity in HFD+H-AP39 and HFD+L-AP39 groups were increased after AP39 intervention,indicating the antioxidant effect of AP39.4.ROS levels can be detected by DHE staining.DHE staining shows that HFD leads to increased ROS production,while AP39 can eliminate ROS fluorescence intensity.AP39 increased the absorbance at 520 nm,suggesting that HFD-induced mitochondrial swelling was improved.MMP is also a mitochondrial membrane potential.In general,MMP can support oxidative phosphorylation in mitochondria and the production of large amounts of ATP.MMP was significantly lower in the HFD group than in the normal diet group.MMP increased significantly after AP39intervention,and the difference was statistically significant.These results showed that compared with HFD-fed rats,The loss of MMP in HFD rats treated with AP39decreased.5.AP39 can enhance mt DNA copy number level,AP39 can significantly down-regulate liver HIF-1αm RNA and reduce HIF-1αprotein expression level.