| The development of society promotes the rapid growth of people’s dietary consumption demand,and the global consumption of meat protein continues to increase,especially red meat.However,there is growing evidence that high consumption of red meat,especially processed red meat,increases the risk of morbidity and mortality from chronic diseases such as nonalcoholic fatty liver disease,type 2 diabetes,cardiovascular disease and all kinds of cancer.In the process of storage,processing and cooking,food proteins such as meat are easily modified by various physical and chemical factors,and in the presence of secondary oxidation products such as sugars and lipids.Oxidation can destroy the structure and biochemical functions of proteins,leading to some sensory and nutritional problems in muscle food,and may have adverse effects on body health.Oxidation of muscle proteins induced by cooking or processing may be an important factor in the harmful effects of red or processed meat intake.However,there are few studies on its biological effects.This subject takes pork as the research object to explore the effects of different processing methods on protein oxidation of pork,as well as the effects of pork intake with different protein oxidation levels on intestinal health and glucolipid metabolism,and its possible mechanism.Firstly,this study used two different methods to cook fresh pork: Sous-vide(SV,70℃vacuum water bath for 1 h)and high temperature and pressure(HTP,121℃,0.2 MPa steam bath for 1 h).The cooked pork was then subjected to in vitro simulated digestion and faecal fermentation of digested products to investigate the effects of processing methods and subsequent digestion processes on the protein oxidation of pork and its in vitro digestion and fermentation characteristics.Then an in vivo evaluation method was established through animal experiments to systematically analyze the effects of pork intake with different protein oxidation levels on intestinal function.The results showed that processing could induce oxidation of pork protein.Compared with SV cooking,HTP significantly increased the oxidation of pork protein before and after slaking,and reduced the in vitro digestive rate of pork protein and antioxidation of digestive juices.Therefore,in subsequent animal experiments,we defined pork cooked under SV as low-oxidative pork(LOP),and pork cooked under HTP as high-oxidative pork(HOP).The results of fecal bacteria fermentation showed that HTP cooking increased the gas production during pork fermentation and the p H value of the fermentation broth,and reduced the production of short-chain fatty acids(SCFAs)in the fermentation broth and the relative abundance of beneficial bacterium Lactobacillus,Bifidobacterium and Coprococcus related to SCFAs production,and elevated the relative abundance of harmful bacterium Escherichia coli.The results of animal experiments showed that HOP intake resulted in the accumulation of oxidative damage markers,increased body weight and plasma endotoxin(LPS)level in mice,decreased SCFAs levels in colon contents,and changed the relative abundance of specific bacterium,including decrease the relative abundance of SCFAs-producing bacterium Lactobacillus,Bifidobacterium and Coprococcus,and elevate the relative abundance of LPSproducing bacterium Escherichia coli,thereby impairing the barrier function of the intestinal tract and inducing oxidative stress and inflammation.Secondly,glucolipid metabolism and thyroid hormone function related indexes were determined in this study to explore the effects of pork intake with different protein oxidation levels on liver lipid metabolism and glucose homeostasis in mice,and its possible mechanism.The results showed that HOP intake increased plasma lipid levels and white fat weight in mice,induced lipid metabolism disorder,and inhibited energy metabolism.HOP intake increased the accumulation of oxidative damage markers and ROS levels in liver,and decreased the antioxidant defense ability and the expression of antioxidant defense-related genes(Nrf2,HO-1 and NQO-1),thereby inducing hepatic oxidative stress.HOP intake increased plasma transaminase(AST and ALT)activities and hepatic proinflammatory cytokine(TNF-α,IL-6and IL-1β)levels,decreased antiinflammatory cytokine(IL-10)level,and up-regulated the expression of genes related to liver inflammation process(TLR4,My D88,NF-k B,TNF-α,IL-6 and IL-1β),thereby inducing hepatic inflammation and impairing liver function.HOP intake elevated liver weight,liver index,liver fat vacuole and fat infiltration area ratio,and liver lipid content,suggesting that HOP caused liver fat deposition.HOP impaired the thyroid function of mice by inducing oxidative stress and inflammatory,which reduced the synthesis and secretion of thyroid hormone and inhibited the body’s energy metabolism.HOP down-regulated the gene and protein expression of the major thyroid hormone receptor β1(TRβ1)in the liver,and reduced the gene expression and activity of type I deiodinase by inducing oxidative stress and inflammation,indicating the peripheral metabolism of thyroid hormone was impaired,thereby reducing the conversion of T4 to active T3,inhibiting liver local energy metabolism and reducing the level of plasma T3.In addition,HOP upregulated the expression of lipid synthesisrelated genes and protein regulated by thyroid hormone in the liver,and downregulated the expression of lipidolysis-related genes and protein.These results suggested that HOP inhibited hepatic energy expenditure by regulating the expression of thyroid hormone target genes and proteins related to lipid metabolism,which led to hepatic fat deposition.In terms of glucose metabolism,HOP elevated fasting blood glucose level in mice,decreased fasting plasma insulin level,and damaged glucose tolerance.HOP induced oxidative stress and inflammation of the pancreas.HOP down-regulated the gene and protein expression of TRβ1 and the gene expression of the thyroid hormone transporter MCT-8 in the pancreas.In addition,HOP downregulated the expression of thyroid hormone-regulated insulin secretion-related genes(Maf A,PDX-1 and GLUT2)and the gene and protein expression of islet β cell antiapoptotic factor Bcl2 in the pancreas,and upregulated the gene and protein expression of proapoptotic factors caspase-3 and Bax,and reduced the quantity of pancreatic β cells.These results indicated that HOP led to pancreatic β cell apoptosis by regulating the expression of thyroid hormone target genes and proteins related to pancreatic function,thereby reducing insulin secretion and increasing the body’s blood glucose level.Pearson correlation analysis showed that pancreaticβ cell apoptosis and insulin secretion-related indicators were significantly correlated with oxidative stress and inflammation,suggesting that oxidative stress and inflammation may be another cause of HOP-mediated low plasma insulin.Dityrosine(Dityr)is a biomarker of protein oxidation and a structural analogue of thyroid hormone T3.In this study,LOP diet and control diet were supplemented with Dityr to the Dityr level in HOP diet to explore the effects of Dityr intake on glycolipid metabolism of mice and its role in HOP-induced metabolic disorders.The results showed that Dityr increased the body weight,white fat weight,liver weight and plasma lipid content of mice,which were closer to those of HOP mice.Dityr led to oxidative stress and inflammatory response in mice,and damaged hepatic function.Dityr damaged thyroid function and peripheral metabolism of thyroid hormone in mice by inducing oxidative stress and inflammation,thus reducing thyroid hormone levels in circulation and peripheral tissues and inhibiting energy metabolism in the body.Dityr down-regulated the gene and protein expression of hepatic TRβ1,and then increased hepatic fat anabolism and decreased fat catabolism by regulating TRβ1-mediated thyroid hormone target genes and proteins expression related to lipid metabolism,thus inhibiting hepatic energy expenditure and leading to hepatic fat deposition.Dityr impaired glucose tolerance and induced hyperglycemia and hypoinsulinemia in mice,which were closer to those of HOP mice.Dityr down-regulated the expression of pancreatic TRβ1 and MCT-8,thereby reducing thyroid hormone transport in the pancreas,and then induced pancreatic β cell apoptosis by regulating the expression of TRβ1-mediated thyroid hormone target genes and proteins related to pancreatic function,thereby reducing insulin secretion and increasing the body’s blood glucose level.These results suggested that Dityr plays an important role in HOPinduced metabolic disorder.Finally,the effects of different processing conditions on the formation of Dityr in pork,fish and chicken were investigated by setting different temperature,time and pressure parameters according to the processing methods commonly used by residents and food industry.The content of Dityr was also determined in some commercially available meat products.The results were as follows: compared with raw meat,processing induced the oxidation of tyrosine in pork and increased the production of Dityr.With the aggravation of processing conditions,the production of Dityr in pork increased gradually,while the content of tyrosine decreased gradually.The overall trend of Dityr production in fish and chicken was similar to that in pork,but its production was significantly lower than that of pork under the same cooking conditions.The reason may be that red meat contains a higher concentrations of heme iron,a pro-oxidant,which accelerates the oxidation of muscle protein compared with white meat.Under the same cooking conditions,the Dityr content measured in chicken is the lowest.Among commercially available meat products,it was the meat floss that had the highest levels of Dityr,which was associated with prolonged cooking and roasting.The addition of sugar further promoted the formation of Dityr.Secondly,canned foods,smoked sausages,Orleans-flavored chicken breasts,fish sausages,pork sausages and pork breasts also contain higher levels of Dityr,which was related to their processing methods.In conclusion,HTP cooking could induce a high level of oxidation of pork protein,while SV reduced the oxidation of pork protein.HOP intake led to the accumulation of oxidative damage markers in the body,decreased the levels of SCFAs in colon contents and increased the level of LPS,thereby impairing the barrier function of the intestinal tract and inducing oxidative stress and inflammatory response.HOP then damaged thyroid function and peripheral metabolism of thyroid hormone in mice by inducing oxidative stress and inflammation,thus reducing the thyroid hormone levels in circulation and liver,and induced hepatic steatosis and hyperglycemia in the body by regulating the expression of thyroid hormone target genes and proteins in liver and pancreas.Dityr plays an important role in HOP-induced metabolic disorder. |
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