| The first part The therapeutic effect of Angiotensin(1-7)in a mouseModel of non-alcoholic steatohepatitisBackgroundNonalcoholic Fatty Liver Disease(NAFLD)is a metabolic stress liver disease closely related to insulin resistance and genetic susceptibility.It is not caused by ethanol and other clear liver damage factors.And it is characterized by bullae steatosis,with or without inflammation,including simple fatty liver,non-alcoholic steatohepatitis(NASH)and cirrhosis.NAFLD is one of important global public health problem of the 21st century.With the change of the Chinese people’s diet and lifestyle,NAFLD is increasing rapidly in recent years.It has become one of common chronic liver disease in China.NAFLD is closely related to hyperlipidemia,obesity and diabetes.Obesity is closely associated with NAFLD.Related studies have shown that obese people are 4.6 times higher risk of NAFLD than ordinary people.NAFLD can occur in all races and the patients of different age groups.According to relevant data,the prevalence of NAFLD was 10%-24%in the general population in Western Europe,the United States and Japan.And the prevalence of NASH was 3%on average,while the prevalence of obese people was as high as 57.5%-74%.The prevalence of NAFLD in developed regions is similar to those of developed countries in China.Clinically,many studies suggest that NAFLD patients not only causes liver disease related to disability and even death,and is closely related to cardiovascular events.NAFLD patients initially characterized by simple fatty liver,a small number of patients may change to NASH.However,NASH is a process between simple fatty liver disease and cirrhosis,portal hypertension,liver tumor.However,the mechanisms in the pathogenesis of NAFLD/NASH is unclear,The NAFLD patients were always told to remove the incentives and causes,and then change the way of life,such as keep a healthy diet,exercise,quit smoking and alcohol to slow down the disease.NASH does harm to many people in recent years.However,the mechanism of the pathogenesis of NAFLD/NASH is unclear,and there are no good treatment for the people of NASH.Therefore,exploring the pathogenesis of NASH,looking for reasonable and effective prevention and treatment measures is of great significance.A "two hits" theory is the important mechanism of the occurrence and development of NAFLD/NASH,including insulin resistance and oxidative stress."First hit" causes lipid droplets deposition in liver cells because of insulin resistance,characterized by simple fatty liver."Second hit" refers to oxidative stress and mitochondrial dysfunction of liver caused by bacterial endotoxin,free fatty acid(FFA),and other harmful factors.Mitochondrial dysfunction may lead to a high level of reactive oxygen species(ROS),inhibition of liver fatty acid oxidation,excessive liver lipid deposition,lipid peroxidation,inflammatory factor release,aseptic inflammation and damage of hepatocyte.It is called NASH clinically.NASH is a local manifestation of metabolic syndrome in the liver,the occurrence of pathological mechanism is more complicated,involving multiple body systems outside the liver.Thus an effective target for the prevention and treatment of NASH must covering multiple body systems,including the liver.Renin angiotensin system(RAS)exist in many body tissues and organs,including cardiovascular system,liver and endocrine system.Therefore,RAS is an appropriate target research for the pathogenesis of NASH.In addition to ACE-AngⅡ-AT1R,Angiotensin-converting enzyme 2(ACE2),angiotensin 1-7(Ang(1-7))and Ang(1-7)receptor Mas are discovered recently.They are new components of RAS.They constitute ACE2-Ang(1-7)-Mas receptor axis.ACE2 and ACE is belong to the same group but their functionality is very different.Angl and AngⅡ were cracked by ACE2 into Ang(1-9)and Ang(1-7)respectively.Ang(1-7)may inhibit many physiological functions of AngⅡ through its receptors Mas,including inhibiting inflammation and antioxidant effect.Therefore,we will further research on this field,in order to further explore the pathogenesis of NASH,provide new train of thought,and improve the clinical treatment strategies.Aims:Methionine-Choline deficient(MCD)feed was used to build a kind of mouse model of NASH.And the mice were injected Ang(1-7)intraperitoneally with the implanted micro-osmotic pump at a rate of 25ug/kg/h for 6 weeks.And we will observe and discuss the pathological features of NASH and the therapeutic effect of Ang(1-7)on NASH.Methods:24 C57BL/6 mice were randomly divided into the following four groups after 1 week adaptive feed:①MCS control group were fed with Methionine-Choline sufficient feed;②MCD group were fed with Methionine-Choline deficient feed.③MCD+Ang(1-7)group were fed with Methionine-Choline deficient feed and injected Ang(1-7)intraperitoneally with the implanted micro-osmotic pump at a rate of 25ug/kg/h.④MCD+NaCl group were fed with Methionine-Choline deficient feed and injected NaCl intraperitoneally.All the mice were kept in a SPF room and recorded the weight once a week.The temperature of the room is 25±2℃.After 6 weeks,all the mice were anesthetized and killed to obtain the serum and liver.Serum alanine aminotransferase(ALT)and aspertate aminotransferase(AST)were detected by automatic biochemical analyzer.Pathological features of the livers were evaluated by haematoxylin and eosin(H&E)stain and oil red O stain.And the overall evaluation of liver fatty change and inflammation for each group were compared with NAFLD Activity Score(NAS).Statistical analysisThe data were analyzed with SPSS 13.0.All of the data are represented as the means±standard deviation.Significant differences between two groups were evaluated by Independent-Samples T Test.P values less than 0.05 were considered statistically significant.Result1、After six weeks,all the mice were alive,compared with the MCS control group,the mice fed with MCD diet move less and had mussy and unpolished hair.And the MCD+Ang(1-7)group is better than MCD group and MCD+NaCl group.The weight of MCS control group begins to increase gradually since the second week while the MCD diet groups begins to decrease gradually.2、Serum ALT and AST.The level of serum ALT and AST between MCD group and MCD+NaCl group is similar,there is no difference between two groups(P>0.05).Both of the two groups are higher than MCS group(P<0.05).Compared with MCD group and MCD+NaCl group,MCD+Ang(1-7)group had a lower level(140.87±60.18;224.55±31.23)in serum ALT,AST.However,there is no difference among them.3,Hepatic pathological changes.Liver gross observation,MCS control mouse liver is deep red and soft.The MCD group and MCD+NaCl group mice liver toughens and the volume significantly decrease.The color of liver turns yellow.However,the mouse livers of Ang(1-7)group is better than MCD group and MCD+NaCl group.H&E staining of mice liver showed that the MCS group is almost normal.However,hepatocyte fatty change,ballooning degeneration,and visible scattered inflammatory lesions were observed among the MCD group and MCD+NaCl group.MCD+Ang(1-7)group is better than the two groups.NAS score of MCD+Ang(1-7)group(2.833±0.7528)is lower than MCD group(5.500±0.5477)and MCD+NNaCl group(4.833±0.7528)(P<0.05).Oil red O staining results also showed that there is no lipid droplets deposition in the livers of MCS group.Lipid droplets deposited in the liver of MCD group and MCD+NaCl group significantly.However,the MCD+Ang(1-7)group(38.33±19.408)is better than MCD group(65.00±10.488)and MCD+NaCl group(68.33±16.021).Conclusion:This research successfully establish a mouse model of non-alcoholic steatohepatitis.And by Ang(1-7)injection intraperitoneally can effectively improve the occurrence and severity of NASH.It provided new targets and train of thought for the treatment of NASH and provided the research model for our next research on the mechanism of therapeutic effect of Ang(1-7).The second part The therapeutic mechanism of Angiotensin(1-7)ina mouse Model of non-alcoholic steatohepatitisBackground and Aims:As is known to all,A "two hits" theory is the important mechanism of the occurrence and development of NAFLD/NASH,including insulin resistance and oxidative stress."First hit" causes lipid droplets deposition in liver cells because of insulin resistance,characterized by simple fatty liver."Second hit" refers to oxidative stress and mitochondrial dysfunction of liver caused by bacterial endotoxin,free fatty acid(FFA),and other harmful factors.Mitochondrial dysfunction may lead to a high level of reactive oxygen species(ROS),inhibition of liver fatty acid oxidation,excessive liver lipid deposition,lipid peroxidation,inflammatory factor release,aseptic inflammation and damage of hepatocyte.It is called NASH clinically.Obviously,the liver oxidative stress and mitochondrial dysfunction are the initiating factor in NASH.NAPDH oxidase(NOX)is the main source of ROS.The NOX family includes NOX1,NOX2,NOX3,NOX 4,NOX 5,DUOX1 and DUOX2.ROS may contribute to the production of ROS.Relevant studies suggest that many cytokines,such as platelet-derived growth factor(PDGF),AngⅡ and lipopolysaccharide(LPS),interleukin 1 beta(IL-1β)and tumor necrosis factor alpha(TNF-α),etc.could stimulate the ROS generation.And then activate the signaling pathways and participate in the regulation of various function.ROS is closely related to cell differentiation and growth,proliferation,apoptosis,and other cellular activities.Intrahepatic ROS may induce aseptic inflammation,this is the pathophysiological basis of NASH.In recent years,many studies suggest that mitochondrial dysfunction and ROS generation closely relate to the activation of NLRP3 inflammasome.NLRP3 inflammasome is a kind of protein complexes,its molecular weight is about 700 kda.Inflammasome,including NLRP3,NLRP1,AIM2 and RIG-Ia,plays an important role in diabetes,atherosclerosis,Alzheimer’s disease,etc.Studies show that IL-1β contributes to insulin resistance,free fatty acid oxidation and liver cell death.The activation of NLRP3 inflammasome is closely related to mitochondrial dysfunction and ROS production.When all kinds of damage happen,it will lead to cell oxidative stress,morphological changes and function disorder of mitochondria,causing increased mitochondrial ROS generation,mitochondrial DNA release.Then it is likely to activate the NLRP3.inflammasome and then lead to a series of damage to liver.Important component of the RAS system including renin,angiotensin converting enzyme(ACE),angiotensinogen,AngⅠ,angiotensin Ⅱ(AngⅡ),angiotensin Ⅱ1 receptor(AT1R)constitutes the classic ACE-AngⅡ-ATR axis.AngⅡ plays a very important role in a series of physiological and pathological function through ATIR receptor.Recent studies show that the RAS system not only exists in the cardiovascular system,but also exists in local tissue such as the liver.It is said that ACE-AngⅡ-ATR axis was motivated among the patients with chronic liver disease.As the most important molecular of RAS system,AngⅡ is closely related to the occurrence and development of the metabolic syndrome and NAFLD.AngⅡ can not only play an important role in blood pressure,blood sugar and blood lipid metabolism but also in the liver injury and liver fibrosis.Relevant research results show that in the liver,AngⅡ can promote the generation of ROS through the activation of NOX4.In recent years,the new components of RAS such as ACE2,Ang(1-7),Mas receptor constitute the ACE2-Ang-(1-7)-Mas axis.It has become another important branch of the RAS,Experts think that ACE2-Ang(1-7)-Mas axis is the negative regulation way of ACE-AngⅡ-AT1R.ACE2 and ACE are belong to the same group but their function is very different.AngⅠ and AngⅡ were cracked by ACE2 into Ang(1-9)and Ang(1-7).respectively.Ang(1-7)may inhibit many physiological functions of AngⅡ through its receptors Mas,including inhibiting inflammation and antioxidant effect.And ACE2 plays a role of antioxidant through Ang(1-7).However,in the NASH mice,whether Ang(1-7)could inhibit the production of ROS and NLRP3 by inhibiting NOX4 so as to inhibit the progress of NASH is unclear.This paper carry out the preliminary study.Methods:1、In vivo,immunohistochemical stain and western blot technique were used to detect NOX4,ASC and Caspase-1 expression in mouse livers of different groups.Transmission electron microscopy(TEM)technique was used to observe the hepatic sinus endothelial cells(SEC)and the changes of the ultrastructure of cell organelles.2、In vitro,Using AML12 cell to the following experiments:(1)Effects of AngⅡon Caspase-1 and ASC were analyzed using Western blot assays.Cells were exposed to AngⅡ(10-7mol/L)for 6,12 and 24 hours.(2)Effects of Ang(1-7)on Caspase-1 were analyzed using Western blot assays.Cells were exposed to Ang(1-7)(10-7mol/L)for 6,12 and 24 hours.(3)Effects of Ang(1-7)(10-7mol/L),AngⅡ(10-7mol/L)and Ang(1-7)+AngⅡ on ROS generation of AML12 cell.Cells were exposed to Ang(1-7),AngⅡ and Ang(1-7)+AngⅡ for 24 hours.Fuorescence intensity was analyzed,reflecting ROS generation.3、The data were analyzed with SPSS 13.0.All of the data are represented as the means±standard deviation.Significant differences between two groups were evaluated by Independent-Samples T Test.P values less than 0.05 were considered statistically significant.Results1、In vivo,immunohistochemical results show that NOX4,ASC and Caspase-1 expression is higher in MCD group and MCD+NaCl group than in MCS group(P<0.05).And compared with MCD group and MCD+NaCl group,NOX4,ASC and Caspase-1 expression of MCD + Ang(1-7)group is lower(P<0.05).The results of western blot is similar with immunohistochemical stain.NOX4 expression is higher in MCD group and MCD+NaCl group than in MCS group(P<0.05).And compared with MCD group and MCD+NaCl group,NOX4 expression of MCD+Ang(1-7)group is lower(P<0.05).The results of Transmission Electron Microscopy(TEM)showed that,compared with the MCS control group,the pores of liver blood sinus endothelial cells reduced in the NASH mice induced by MCD diet.And swollen and deformed mitochondria were observed obviously in the hepatocyte of NASH mice.However,the pores of liver blood sinus endothelial cells and mitochondria is improved in MCD+Ang(1-7)group.2、In vitro,AngⅡ increase the expression of Caspase-1 and the time of the most effect is at 24h;Ang(1-7)decrease the expression of Caspase-1 and the time of the most effect is at 24h.Further,AngⅡ increase the ROS generation of AML12 cell,while Ang(1-7)decrease the ROS generation.Conclusion:As we can see from the results of the first part,compared with MCD group and MCD+NaCl group,Ang(1-7)could improve the severity of NASH.The results of TEM showed that Ang(1-7)can inhibit the reduction of liver blood sinus endothelial and prevent the mitochondrial from swelling and deforming.This suggests that Ang(1-7)could maintain the stability of mitochondrial morphology and function.In addition,the results of immunohistochemical stain and western blot showed that NOX4,ASC and Caspase-1 expression in MCD group and MCD+NaCl group is higher than MCS group,while NOX4,ASC and Caspase-1 expression of MCD+Ang(1-7)group is lower than MCD group and MCD+NaCl group.In addition,Ang(1-7)decrease the ROS generation of AML12 cell.These results suggest that Ang(1-7)could inhibit the expression of NOX4 and the activation of NLRP3,then inhibit the progress of NASH in mice. |
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