| Non-alcoholic steatohepatitis(nonalcoholic steatohepatitis,NASH)is an acquired metabolic disease closely associated with insulin resistance and genetic susceptibility.It is a pathological stage in the development of simple fatty liver into hepatic fibrosis.It is one of the important causes of cryptogenic cirrhosis,and is common in patients with obesity,type 2 diabetes mellitus,hyperlipidemia and other metabolic syndrome.Follow-up studies have shown that the prognosis of NASH is poor,with about 50%of NASH patients developing liver fibrosis within 6 years,10-15%of patients developing cirrhosis within 10 to 20 years,and 9%of patients developing liver fibrosis within 6 years.In the past 20 years,a large number of pathological studies on NASH and clinical trials to treat NASH have been carried out worldwide,which has led to an increasing understanding of the disease.However,the pathogenesis of NASH is still not very clear,although it is generally perceived that the interaction between genetic susceptibility and multiple metabolic disorders may be the main cause of NASH.Furthermore,there are still no effective treatments and drugs for NASH.Currently.the most common treatments include lifestyle interventions,such as diet control and increased exercise,medication and surgery,which showed some therapeutic benefits for some NASH patients.For example,when the body weight loss is more than 9%,the patient's hepatic histology can be improved obviously.However,these treatments showed limited efficacy and cannot stop the overall rising trend of NASH incidences.As a result,more effective drugs and therapeutic methods for NASH are in urgent demand.Three main research work were included in this thesis:Part 1.Defining RLA8 as a NASH drug candidate with both PPARs-?/?/?and GPR40 tetra-agonist activityPeroxisome proliferator-activated receptor(PPAR)is a nuclear receptor which plays a key role in regulating metabolic homeostasis,immune inflammation and cell differentiation.Studies in rodents and in human clinical trials have shown that PPAR agonists could benifit NASH treatment through a variety of mechanisms,including the induction of genes involved in(3 oxidation,reducing inflammation,reducing oxidative stress,and increasing the secretion of beneficial adipokines such as adiponectin.G protein-coupled receptor 40(GPR40),also known as free fatty acid receptor 1(FFAR1),is a seven-transmembrane domain receptor that is mainly expressed in pancreatic ? cells.It couples with the G protein ?-subunit of the Gq family to regulate the physiological process of cells.For example,GPR40 can increase the Gq protein-mediated phospholipase C(PLC)activity and intracellular calcium level in islets.Therefore,it is responsible for glucose-dependent stimuli of insulin secretion.Recent studies have confirmed that GPR40 is also expressed in hepatocytes and has insulin sensitizing effect.In high-fat diet(HFD)-induced diabetic mice,activation of GPR40 reduces blood glucose,lowers plasma insulin and improves glucose intolerance and insulin resistance.The efficacy of GPR40 agonists to treat diabetes and their safety have been demonstrated in patients with type 2 diabetes.However,even though PPARa agonists appear to be effective in animal studies,they are not effective in clinical trials.PPAR? agonists may also be promising,but data from human trials are very limited.PPARy agonists are effective in improving histological characteristics,including fibrosis,in clinical trials.However,widespread use of PPARy agonists such as thnazolidinediones is limited by their side effects.As mentioned above,GPR40 agonists are highly effective in diabetic patients,but little research has been done about their efficacy to improve hepatic steatosis.Therefore.agonists capable of activating more than one target of glycolipid metabolism are worthy of study.In previous studies,our group designed and synthesized a series of compounds based on a natural product platform,resveratrol.and found that a compound ZBH could activate all three subsclasses of PPARs and significantly reduce the level of blood lipid in hyperlipidemia mice.However,its activity needs to be further improved.In this research,a series of compounds combining the structural characteristics of ZBH and current new NASH drugs were screened,and 10 compounds were found to possess better activity than ZBH.Further in vitro studies by cell receptor activation assays and in vivo studies using an acute hyperlipidemia mouse model revealed that compound RLA8 "was a quadruplet agonist for PPARs-?/?/? and GPR40 and had good lipid-lowering activity.Therefore,we considered RLA8 as a promising drug candidate for NASH and performed additional detailed studies on its efficacy to treat NASH using animal models.Part 2.Evaluation of the efficacy of RLA8 in the treatment of NASH with two NASH mouse models induced by methionine/choline deficiency diet(MCD)and high fat diet(HFD)The ideal animal models for such studies should encompass all of the features of human NASH,including obesity.insulin resistance,steatohepatitis and fibrosis However,there is currently no single animal model that can meet these requirements In the past few decades,many NASH animal models have been developed,among which dietary models are considered the best.In particular,the high fat diet(HFD)model can well replicate changes in metabolic parameters observed in human fatty liver disease,such as obesity,elevated glucose and insulin resistance,but its liver pathological characteristics are not sever;for example,it is difficult to induce fibrosis The methionine choline-deficient diet(MCD)model can replicate human NASH histological features such as steatohepatitis and fibrosis and cause more severe inflammation,oxidative stress,and cell death and fibrosis.Therefore,it is suitable as a histologically advanced NASH model.However,the MCD model has symptoms that are opposite to human metabolic characteristics,such as weight loss and no insulin resistanceTaking the pros and cons of each animal model into consideration,both HFD and MCD models were used to evaluate the efficacy of RLA8 to treat NASH.The MCD animal model was also used to explore the possible mechanisms of action of RLA8.Furthermore,resveratrol(RSV)and obecholic acid(OCA),the only clinically approved adjuvant therapy for NASH,were employed as positive controlsFirst,the influence of RLA8 on body weight and food intake in MCD and HFD mice was investigated.It was shown that as compared to that of nagtive control(NC)group mice,the body weights of MCD mice in the model control(MC)group decreased significantly,which lasted until the end of the experiments,indicating the occurrence and development of NASH symptoms.After RLA8 treatment,the body weight loss was halted,and a statistically significant difference in the body weight between RLA8 and MC groups was observed one week after RLA8 administration(P<0.05).RSV and OCA treatments showed a similar impact on mouse body weight but their difference from the MC group was not statistically significant.These results indicated that RLA had better or at least similar activities to inhibit body weight loss in MCD mice,as compared to positive controls RSV and OCA.In contrast to the MCD model.HFD caused a significant increase in body weight of the MC mice as compared to NC mice(P<0.01).RLA8 and OCA treatments had similar effects to invert excessive body weight gain.Their difference from the MC group was statistically very significant(P<0.01).RSV treatment could also stop mice from gaining more body weight and its difference from the MC group was statistically significant(P<0.05).However,the efficacy of RSV was not as high as that of RLA8 and OCA.In addition,all three compounds did not show a significant impact on food intake by mice of both modelsAfter the mice in above experiments were autanized,their livers were extracted and subjected to histochemical and histopathological examinations.H&E staining of liver tissues revealed remarkable hypertrophy of hepatocytes in MC mice of both MCD and HFD models as compared to NC mice on normal diets.Oil red staining of the liver tissues showed that the mice in both MC groups had severe steatohepatitis and a remarkable hepatic lipid accumulation as compared to NC mice.In RSV treatment groups,neither MCD nor HFD mice showed significant histopathological improvement compared to MC mice.In contrast.OCA and RLA8 treatments reversed both MCD and HFD-induced NASH and could significantly lower the number and amount of fat droplets formed in the livers.It was also noticed that the number and size of inflammatory foci were significantly reduced in OCA and RLA8-treated mice Furthermore,the therapeutic efficacy of RLA8 was notably better than that of OCA In fact,the morphological looks of liver tissues from RLA8-treated mice were rather similar to that of normal mice.To facilitate quantitative analysis and comparison of the results,we assigned to the tissues in each group an NAFLD activity score(NAS)according to the criteria.Clearly,the NASs of RLA8 group mice for both NASH models were significantly lower than that of the MC group,a reduction of 53.1 and 60.2%(P<0.01)for the MCD and HFD model,respectively.These results further corroborated that RLA8 could significantly improve liver injuries caused by NASH and its efficacy was slight better than OCA(NASs 3.3 and 2.3 vs.4.3 and 2.7).To gain more insights into related pathology,we also performed biochemical analysis of the mice.In accordance with the observations above,we found that the contents of hepatic TG,TC,and FFA in MC mice of MCD model increased by 8.75,4.30,and 1.61 folds,respectively,as compared to that in NC mice,and they increased by 2.5,3.05,and 1.43 folds in HFD MC mice.Similarly,the hepatic lipoperoxide content in MC mice of both models also exhibited a significant increase.The accumulation of TG,TC,FFA,and lipoperoxide in mouse livers was associated with hepatic dysfunction as revealed by the significantly elevated serum ALT for both models.The results further showed that RLA8 treatment could reverse hepatic lipid accumulation in both NASH models.Compared to that in MC mice,hepatic TG,TC,and FFA contents in RLA8-treated mice were reduced by 61.1%.40.1%,and 38.4%in the MCD model and by 34.8%,52.2%,and 29.2%in the HFD model,respectively.OCA treatment gave similar results,but it reduced hepatic TG,TC,and FFA contents by only 21.3%,28.4%,and 7.9%in the MCD model and by 14.9%.34.7%,and 20.4%in the HFD model,which were much less impressive than the reductions induced by RLA8.On the other hand,RSV could only marginally reduce FFA accumulation and had no significant impact on hepatic TG and TC contents.Moreover.RLA8 treatment significantly increased the HDL-C content(P<0.01),but OCA and RSV did not have any obvious effect.Although RSV treatment had no significant influence on hepatic TG and TC contents,it could reverse the lipoperoxide content of NASH mice to the normal level,just like RLA8 and OCA.In addition,all three compounds were able to restore hepatic functions in NASH mice,as indicated by the recovered serum ALT levels after treatments.Again,RLA8 seemed to be more potent than OCA and RSV in this respect.These results agreed very well with that of above histochemical and histopathological studies,which indicated that RLA8 was more potent than OCA,which in turn was much more potent than RSV,to reverse hepatic lipid accumulation and steatohepatitis in both disease models.NASH patients are usually accompanied with metabolic inflammation featured by elevated levels of C-reactive proteins and various interleukins(IL).In consistent with these observations,we also noticed an elevated level of inflammatory activities in the MCD and HFD mice,including an increased number of inflammatory cells and elevated level of major hepatic inflammation markers,such as TNF-a.IL-6 and IL-10 It was revealed that RSV.OCA.and RLA8 could all reduce NASH-induced TNF-a.IL-6,and IL-10 secretion and,consequently,alleviate related hepatic inflammation.Again,RLA8 showed more potent therapeutic efficacy than RSV and OCA,especially in the more severe MCD NASH model.Hepatic fibrosis is triggered by chronic injuries,especially inflammation,and represents one of the common and severe concerns in NASH,since it can lead to portal hypertension and cirrhosis to disrupt hepatic function.Therefore,we also examined the impact of RLA8 treatment on hepatic fibrosis based on liver biopsy.In this regard,the liver sections from MCD and NC mice were stained with trichrome and then subjected to histopathological examination.In line with previous reports,a devoid of essential amino acid methionine and choline in MCD diet promoted severe steatohepatitis and the occurrence of appreciable hepatic sinusoidal fibrosis.Evidently,RSV,OCA,and RLA8 treatments could all alleviate hepatic fibrosis.In particular.OCA and RLA8 treatments could reverse hepatic fibrosis almost completely.In agreement with this discovery,elevated expression of the hepatic collagen al gene in MCD mice was effectively suppressed by all three compoundsInsulin resistance is another important hallmark for NAFLD.However,MCD does not cause insulin resistance,although it can induce a more severe form of NASH and fibrosis in mice.In contrast,HFD can induce mild peripheral insulin resistance,as reflected by significantly increased serum levels of glucose,insulin,TG and TC,as well as HOMA-IR,in HFD MC mice.Consequently,we examined the impacts of RSV,OCA.and RLA8 treatments on insulin resistance associated with the HFD model.It was found that all three compounds led to a significant reduction in serum glucose and insulin levels and HOMA-IR,suggesting great improvement in insulin resistance in these mice.This was associated with the significant improvement of HFD-induced hyperlipidemia,namely,elevated serum TG and TC levels,in the case of OCA and RLA8(P<0.05),although changes in serum TG and TC levels in the RSV group were not so obviousPart 3.Analysis of potential pharmacological mechanisms of RLA8 in the treatment of NASHTo help elucidate the underlying mechanism by which RLA8 improves NASH in MCD mice,we analyzed the expression level of PPAR-related genes and proteins involved in hepatic lipid metabolism,inflammation,and fibrosis by PCR and western blot.The expression of genes and proteins regulating lipid synthesis,including SREBP1,FAS and SCD1,or inflammation and fibrosis,including VCAM1,TIMP1 and TGF?1 were dramatically upregulated in MC mice,as compared to NC mice.RLA8 treatment was found to significantly suppress the expression of all assessed genes and proteins,except for TGF?1.These results suggested that RLA8-induced reduction in hepatic lipid accumulation,inflammation and fibrosis was coupled with reduced expression of the downstream hepatic genes and proteins of PPARs Considering that the SREBP1,FAS and SCD1 expression is regulated by the AMPK-ACC pathway and that the AMPK-ACC pathway is closely related to fatty acid biosynthesis and lipogenesis,we then assessed the AMPK activity by measuring Thr172 phosphorylation of AMPKa and its major downstream protein p-ACC(Ser79),as AMPK activation can lead to the phosphorylation and inhibition of ACC,which is the rate-limiting enzyme in fatty acid biosynthesis.Their western blot results disclosed that,compared to the MC group.RLA8 treatment could significantly enhance the phosphorylation of AMPKa and ACC.In conclusion,we have introduced in this work a completely new class of anti-NASH drug,such as RLA8.It was proved to be a balanced quadruple agonist for PPARs-?/?/? and GPR40 and exhibited a superb efficacy to reverse NASH-induced hepatic lipid accumulation,steatosis,inflammation,fibrosis,and other damages associated with both HFD-induced mild and MCD-induced severe mouse NASH models.Most impressively,RLA8 showed significantly better efficacy to treat NASH than reference "drugs" RSV and OCA,whereas currently OCA is the most promising NASH druy candidate that is in Phase III clinical trials and there has been no clinically approved drug to treat NASH yet.In addition,our preliminary studies also showed that RLA8 had fewer and less severe side effects than the reference drugs.Therefore.RLA8 represents a new NASH drug candidate worthy further investigation and we are now in the process to push it towards clinical studies. |
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