Maternal Betaine Supplementation Attenuates NAFLD By NLRP3 Inflammasome Pathway In Offspring Mice

Background:The prevalence of Non-alcoholic fatty liver disease(NAFLD),one of the most common chronic liver diseases in adults,has increased in children and adolescents in recent years,especially in those with adiposity.This phenomenon is not only linked to poor diets in modern life,but also to maternal obesity during pregnancy or breastfeeding.Studies have shown that maternal overweight and obesity are associated with the development of NAFLD in children.Betaine is an essential nutrient for human body,and the severity of NAFLD is related to the betaine-deficiency in adults.Previous studies have confirmed the effects and effectiveness of betaine as a methyl donor in NAFLD.Additionally,the level of betaine in plasma dropped during pregnancy.If betaine intervention can be carried out in mothers during pregnancy,preventing the occurrence and development of fetal NAFLD,it will provide a new idea for the control of the disease in the early stage.NAFLD is strongly associated with obesity,which is associated with chronic inflammation.Obesity-induced local and systemic inflammatory states may affect the liver and may lead to elevated levels of inflammation in the placenta and fetus during pregnancy.NLRP3 inflammasome plays a vital role in the occurrence and development of NAFLD.Inhibition of the activation of NLRP3 inflammasome can alleviate the steatosis symptoms of NAFLD.Of note,there are few studies on the effect of betaine on inflammatory response and whether it can inhibit the activation of NLRP3 inflammasome remains unknown.Therefore,we investigated the effect of betaine on NAFLD in offspring by supplementing maternal betaine during pregnancy and lactation.Meanwhile,the expression of NLRP3 inflammasome related genes and their downstream genes were studied to explore whether the alleviating effect of betaine on progeny NAFLD was related to its correction effect on the abnormal expression of NLRP3 inflammasome related genes.Methods:(1)C57BL/6 female mice were completely randomly divided into 4 groups:WILD,NAFLD,NAFLD+1%BET and NAFLD+2%BET.STZ was injected three times per 24 hours for NAFLD modeling after pregnancy.A high-fat diet was given after successful modeling.Betaine is configured in 1% and 2% aqueous solutions.Offspring were also divided into 4 groups according to the intervention:WILD,FNAFLD,1%BET and 2%BET.After weaning,the offspring were given normal diet and drinking water for 8 weeks and then dissected to measure relevant indexes.(2)The levels of ALT,AST,TG,TC,LDL,HDL and GLU in serum were detected by automatic biochemical analyzer.HE staining and Oil Red O staining were used to observe the histopathological changes of mice liver.GPO-PAP method was used to measure the level of TG in mice liver.(3)The serum levels of TNFα and IL-6 were measured by ELISA.(4)RT-PCR was used to detect the level of m RNA transcript of TNFα,IL-6,NLRP3,ASC,CASP1,IL-1β,IL-18,DNMT1,DNMT3 A and DNMT3 B.The expression of NLRP3,ASC,CASP1,IL-1β,IL-18 protein was detected by Western blot.Determined the methylation level of specific genes by sequencing after bisulfite modification.(5)Determined the global DNA methylation level by colorimetry.The levels of betaine,choline,trimethylamine oxide(TMAO),homocysteine(HCY),Sadenosylmethionine(SAM)and S-adenosylhomocysteine(SAH)in mice liver were determined by HPLC.Results:(1)The serum ALT of F-NAFLD group was significantly increased,but it decreased after 1% betaine and 2% betaine intervention.Compared with the F-NAFLD group,the TG content in the liver tissues of offspring in 1%BET and 2%BET groups was significantly decreased.(2)The m RNA relative expression levels of CASP1 and IL-18 in F-NAFLD group were significantly increased while the m RNA relative expression levels of CASP1,IL-1β and IL-18 in 1%BET group were decreased,and the m RNA relative expression levels of NLRP3,ASC,IL-1β and IL-18 in 2%BET group were decreased.(3)The results of protein expression were similar to those of gene expression.(4)The methylation level at-2326 and-2259 of NLRP3 gene in the 2%BET group was significantly lower than that in the F-NAFLD group.The methylation level of ASC gene promoter region in young mice was not different at each site.(5)No significant difference was found in global DNA methylation level,liver betaine,choline and TMAO levels.Liver Hcy in 1%BET group was significantly higher than that in FNAFLD group,and significantly reduce liver SAH level in 1%BET group and 2%BET group.The ratio of SAM/SAH in the liver remain unchanged in each group.(6)Compared with F-NAFLD group,DNMT1 gene and DNMT3 B m RNA levels were significantly decreased in 2%BET group.For DNMT3 A gene,the relative m RNA expression of this gene was decreased in both 1%BET and 2%BET groups,and the difference was statistically significant.Conclusion:(1)Maternal betaine intervention alleviated visceral fat accumulation and liver cells damage in offspring with NAFLD induced by high fat diet +STZ.(2)Maternal betaine supplementation regulated the abnormal expression of NLRP3 inflammasome related genes induced by fetal-derived NAFLD.Maternal betaine downregulated the expression of NLRP3,ASC,CASP1,and then down-regulated the expression of IL-1β and IL-18 genes.The result of NLRP3 inflammasome protein expression was similar to that of gene expression.These results suggested that maternal betaine supplementation may reduce NAFLD in offspring by alleviating inflammatory response.(3)Maternal betaine intervention might not related to the methylation level of DNA promoter region of NLRP3 inflammasome related genes.(4)Maternal betaine supplementation also affected the metabolism of methyl donors in the liver of offspring mice to a certain extent.After maternal betaine intervention,liver Hcy and SAH levels decreased in offspring with NAFLD.