Association Of Gender Disparity And MyD88-dependent IL-6Signaling Pathway In Nonalcoholic Steatohepatitis

Part1Association of gender disparity and MyD88-dependent IL-6signalingpathway in nonalcoholic steatohepatitis induced byMCD diet in C57BL/6miceObjective: NASH was modeled in C57BL/6mice by feeding MCD diet. Basedon the observation of gender disparity in this model, association of sex differencesand MyD88-depented IL-6signaling pathway was analyzed.Methods：Twenty-six14-week-old C57BL/6male mice were randomly dividedinto two groups:(1)male control group with MCD control diet feeding,(2)male MCDgroup fed on MCD diet. Twenty-six14-week-old C57BL/6female mice were alsorandomly divided into two groups:(1)female control group with MCD control dietfeeding,(2)female MCD group fed on MCD diet.All mice sacrificed at the end of4weeks. Serum concentrations of alanine aminotransferase (ALT),aspartateaminotransferase (AST) and γ-Glutamyltransferase(γ-GT) were determined by usingan automatic biochemical analyzer. In addition, portions of left hepatic lobe from allmice were fixed. And tissue sections were stained with hematoxylin-eosin(H&E),osmic acid to examine the histopathology, which was done double-blind by twopersons. Furthermore, the nonalcoholic fatty liver disease activity score (NAS) wasrecorded.Then,the mRNA level of MyD88and IL-6in the liver was analyzed by usingreal time fluorescence quantitative polymerase chain reaction (qRT-PCR).Finally, theprotein levels of MyD88and IL-6in the liver were determined with the help ofWestern blot analysis.Results:Both male and female mice that were fed on MCD diet exhibited steatosis and inflammation of the liver. Fatty change was mostly of a macrovesiculartype, involving all zones of the hepatic acinus. The inflammatory infiltrate waspredominantly composed of foci of lymphocytes randomly distributed in the portaland lobule. The inflammatory infiltrate was evident and ballooning degeneration ofhepatocytes was observed in male mice that were fed on MCD diet. However,therewere less inflammatory infiltratewas and no hepatocyte ballooning detected in femalemice that were fed on MCD diet. No steatosis or inflammation was seen in the controldiet-fed miceCompared with the control groups, male and female mice feeding on MCD dietshowed a significant higher in steatosis score, inflammatory score,NAS and levels ofserum ALT,AST, γ-GT(P＜0.05）. We found no big differences in the steatosis scorefor male MCD group in contrast with the female ones (P>0.05), while the lobularinflammation score,NAS and the levels of serum ALT,AST, γ-GT were significantlyhigher in males than in females (P<0.05), especially with the predominant rise on theaspects of ALT&AST.Compared with male and female control groups respectively, male and femaleMCD groups were witnessed to have a significant increase in the mRNA and proteinlevels of MyD88, IL-6in the liver (P<0.05). The mRNA and protein levels ofMyD88,IL-6in the liver were greatly elevated in the male MCD group relative to thefemale MCD group（P＜0.05）.Conclusion: NASH induced by MCD diet in male mice were much more seriousthan female mice, which is displayed as more serious inflammatory cell infiltration,and hepaticytes ballooning degeneration, as well as higher histopathology scores.Likewise, serum biochemical change in male mice was higher than female mice.Furthermore, serum transaminases, including both ALT and AST rose significantlyhigher than γ-GT. As for male C57BL/6mice fed on MCD diet，the express level ofMyD88,IL-6mRNA and protein in liver were significantly higher than female mice,which suggests gender disparity of NASH induced by MCD diet has links with theactivation of MyD88-dependent IL-6signaling pathway. Part2Effect of sex hormone-specific intervention on nonalcoholic steatohepatitisinduced by MCD diet and its association with MyD88-dependentIL-6signaling pathwayObjective: NASH was modeled in C57BL/6male mice by feeding on MCD diet.Male mice were subjected to sex hormone-related interventions such as orchidectomy,and orchidectomy combined with administration sex hormone, which was aimed atinvestigating the effects of sex hormone-specific intervention on pathology andprogression of NASH, and also on the MyD88-dependent IL-6signaling pathway aswell.Methods:Sixty-five14-week-old C57BL/6male mice were randomly divided intofive groups:(1)male control group with MCD control diet feeding,(2)male MCDgroup fed on MCD diet,(3) orchidectomy MCD group fed on MCD diet and subjectedto orchidectomy,(4) orchidectomy MCD+androgen group fed on MCD diet andsubjected to orchidectomy plus with subcutaneous injection of testosterone propionate(5mg/kg/d),and(5) orchidectomy MCD+estrogen group fed on MCD diet andsubjected to orchidectomy plus with subcutaneous injection of estradiol benzoate(0.5mg/kg/d). All mice sacrificed at the end of4weeks. The concentration of estradioland testosterone were measure by ELISA. For the same purpose, serumconcentrations of ALT, AST and γ-GT were determined by using an automaticbiochemical analyzer. Moreover, portions of left hepatic lobe were fixed. Also,tissuesections were stained with H&E and osmic acid to examine the histopathology. AndNAS was recorded. Then, the mRNA level of MyD88and IL-6in the liver wasanalyzed by means of qRT-PCR. At last,the protein levels of MyD88and IL-6in theliver were determined by using Western blot analysis.Results: It was proved that there were significantly lower serum levels oftestosterone in male mice models of MCD-induced NASH subjected to orchidectomy（P＜0.05）. After the mice were treated with exogenous testosterone propionate, theserum testosterone level was significantly elevated (P＜0.05）and then back to thelevel without surgery. However, when exogenous estradiol benzoate was administered to orchidectomy-treated male NASH mice, there was a significant increase in serumestradiol level.There illustrated milder fatty change, inflammatory infiltrate and ballooningdegeneration of hepatocytes, lower NAS, lower ALT and AST in the surgicallytreated mice, compared with untreated mice (P<0.05). And the level of γ-GT wasslimly reduced (P>0.05). It was found out that surgically treated pius withtestosterone propionate mice had a lot of steatosis, accompanied with plenty ofinflammatory infiltrate and occasional appearance of ballooning degeneration ofhepatocytes of the liver. Compared with orchidectomy and no surgical mice, nosignificant changes in NAS, ALT, AST, and γ-GT were observed (P>0.05). However,when orchidectomy-treated male NASH mice were administered to exogenousestradiol benzoate, partial fatty change could be tested, with a small amount ofinflammatory infiltrate and no evidence of ballooning. NAS, ALT, and AST levelbecame apparently reduced, in comparison with the other three groups (male MCDmice, male MCD+orchidectomy mice, and male MCD+orchidectomy+testosteronepropionate mice)(P<0.05). On the other hand, only compared with male MCD mice,there existed a significant difference inγ-GT.The level of MyD88，IL-6mRNA and protein decreased significantly in NASHmodel mice4weeks following orchidectomy, when compared with untreated maleMCD-diet mice (P<0.05) Combining orchidectomy with administration of exogenoustestosterone propionate did not change the mRNA and protein level of MyD88andIL-6when compared with the surgically treated and untreated MCD-diet male mice(P>0.05). However, when surgical treatment was supplemented with exogenousestradiol benzoate, we detected a significant decrease in MyD88and IL-6mRNA andprotein level in comparison with the other three groups (male MCD mice, maleMCD+orchidectomy mice, and male MCD+orchidectomy+testosterone propionatemice)(P<0.05).Conclusion: The pathology damage of nonalcoholic steatohepatitis which induced byMCD diet in male C57BL/6mice, was significantly improved after the mice were treated with orchidectomy or orchidectomy plus with estradiol. In particular, steatosisand inflammatory infiltrate was the slightest and NAS was lowest in group whichusing orchidectomy plus estrogen supplement. Serum biochemical change was cutdown by orchidectomy or estrogen supplement. And the level of serum ALT and ASTwere significantly reduced. There were no significantly change in γ-GT, whenorchidectomy were carried. However, level of γ-GT dropped down obviously, whilemale mice treated with orchidectomy in combination with estrogen supplement. ThemRNA and protein level of MyD88, IL-6was down regulated by either orchidectomyor estrogen supplement. In particular, The combined application of orchidectomy andestrogen enhanced this effects. This suggests that sex hormone-specific interventionimprove liver injury in NASH in male C57BL/6mice fed on MCD diet,due toinhibition of MyD88-dependent IL-6signaling pathway.