Exploring The Mechanism Of Resveratrol Intervening On Mouse Liver Fibrosis Induced By Low Dosage Inorganic Mercury Exposure With Long-Term Based On The Liver-Gut Axis

Mercury is a toxic heavy metal.Mercury pollution caused by its wide application in production and life has become a serious environmental problem.Mercury intake through food,drinking water,and air may cause toxic effects on the body,especially the liver,and induce liver fibrosis and other diseases of livestock and poultry.The value will be decline or elimination of livestock and poultry because of mercury intake and then cause serious economic losses.In addition,the enrichment effect of mercury in the food chain is very significant,and long-term low dose mercury exposure will lead to animal source food safety problems.Resveratrol（Res）is a natural polyphenol which is widely found in plants,including grapes,berries,and nuts,which has important health benefits due to the activities of antioxidant,antitumor,anti-inflammatory,and immunity regulation.Therefore,it is of great practical significance to study the mechanism of liver fibrosis induced by long-term and low dose inorganic mercury exposure in mice and the preventive and therapeutic effects of Res.The trial was divided into 3 parts.Part 1 Trial:Long-term and low-dose inorganic mercury exposure mouse model establishment.Twenty-eight Kunming mice were randomly divided into 4groups of 7 in each group.The control group drank normal water.The low,medium,and high dose groups were given an aqueous solution containing 25,50,and 100 mg/L Hg Cl₂.Mice eat freely.The trial period was 24 weeks.The ileum and the contents were collected for 16S r RNA sequencing.Mice serum was collected for liver function biochemical indexes,blood alcohol concentration（BAC）,lipopolysaccharide（LPS）concentration and serum metabolomics detection.Liver tissues were collected for histopathological testing（HE staining,Masson staining and oil red O staining）,oxidative stress indexes,hydroxyproline（HYP）concentration detection,transcriptomics detection,real-time quantitative reverse transcription-polymerase chain reaction（RT-q PCR）and western blot detection.Ileal tissue was collected for HE,RT-q PCR,and western blot detection.The results showed that:（1）The results of 16S r RNA sequencing of mouse ileal contents showed that inorganic mercury induced intestinal flora disorders in mice.Inorganic mercury exposure induced the relative abundance reduced of Corynebacterium,Dietzia,Glutamicibacter,Bifidobacterium,Enterococcus,and Brachybacterium（P<0.05）,and the relative abundance increased of Lachnospiraceae＿NK4A136＿group,Clostridium＿sensu＿stricto＿1,Romboutsia,Ruminococcus,Eubacterium＿coprostanoligenes＿group,Eubacterium＿xylanophilum＿group,Spireospiaceae UCG-005,Spireospiaceae NK4A214＿group,Colidextribacter,and Helicobacter（P<0.05）,which may be associated with intestinal barrier damage,decreased spermidine production,and elevated endogenous ethanol.（2）Serum metabolomic results showed that inorganic mercury exposure affected methionine metabolism,steroid biosynthesis,and bile acid biosynthesis,and the key metabolites is the decrease levels of 4-Aminobutyraldehyde,acetylcysteine,allantoin,lathosterol,and spermidine.（3）Biochemical results showed that inorganic mercury exposure induced increased serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）activities in mice.（4）Inorganic mercury exposure induced elevated BAC,indicating that inorganic mercury induced endogenous ethanol metabolism disorders in mice.（5）Increased serum LPS concentration indicates that the intestinal barrier of mice is damaged,and intestinal bacterial products enter the body.（6）The results of liver pathological changes showed that different doses of inorganic mercury exposure induced liver lipid accumulation and fibrosis and other liver damage in mice.（7）Inorganic mercury induced the increase in malondialdehyde（MDA）levels,reduced glutathione（GSH）concentration and superoxide dismutase（SOD）activity in the mice liver,indicating that inorganic mercury exposure induced oxidative stress in the liver.（8）Inorganic mercury exposure induced increased liver HYP levels,indicating that inorganic mercury exposure induced liver fibrosis.（9）RT-q PCR and western blot of ileal tissue results showed that inorganic mercury exposure induced mouse ileal sealant protein occludin（Ocln）,cadherin 1（Cdh1）,claudin（Cldn1）,and tight junction protein 1（TJP1）significantly reduced of m RNA expressions and protein levels（P<0.05）,indicating the intestinal barrier damage in inorganic mercury-exposed mice.（10）Hepatic transcriptomics,RT-q PCR and western blot results showed that inorganic mercury exposure induced abnormal cholesterol and bile acid metabolism in the liver of mice[m RNA expressions and protein levels were significantly reduced of 3-hydroxy 3-methylglutaryl Co A reductase（Hmgcr）,cholesterol 7alpha hydroxylase（Cyp7a1）,aldehyde keto reductase family 1,member B7,cholesterol 24α7hydroxylase（Cyp39a1）,and cysteine sulfinic acid decarboxylase（P<0.05）],abnormal glucose metabolism[m RNA expressions and protein levels were significantly reduced of the peroxisome proliferative activated receptorγ,coactivator 1 alpha（PGC-1α）,glucose-6-phosphatase,catalytic subunit（G6pc）,and pyruvate dehydrogenase kinase isoenzyme 4（Pdk4）（P<0.05）],oxidative stress[m RNA expression and protein level was significantly decreased of glutathione peroxidase 3（Gpx3）,and increased of the cytochrome P450,family2,subfamily E,polypeptide1（Cyp2e1）（P<0.05）],inflammation[increased m RNA expressions and/or protein levels of serum amyloid A1protein（Saa1）,serum amyloid A2 protein,interferon induced protein 44 like,and chemokine（C-X-C motif）ligand 11（P<0.05）],abnormal cell cycle[m RNA expressions and protein levels were significantly reduced of cyclin-dependent kinase inhibitor 1A（Cdkn1a）,cyclin-dependent kinase inhibitor 2C,growthβarrest and DNA-damage-inducible beta and gamma（P<0.05）,and increased in cyclin D1（Ccnd1）and 2（P<0.05）],and liver fibrosis[m RNA expressions and protein levels were significantly reduced of Cdh1,retinol binding protein1（Rbp1）,and glutathione S-transferase,pi 2（P<0.05）].Part 2 Trial:Res intervention in mice for inorganic mercury poisoning test.Twenty-eight Kunming mice were randomly divided into 4 groups of 7 in each group.The control group drank water normally.The Hg group was given with drinking water containing 100 mg/L of Hg Cl₂.The Hg+Res group was given with drinking water containing 100 mg/L of Hg Cl₂,and gavage Res 100mg/kg body weight.Res group was given normal drinking water,and gavage Res 100 mg/kg body weight.Mice eat freely.The trial period was 24 weeks.Mouse serum was collected for liver function biochemical indexes,BAC and LPS concentrations.Liver tissues were collected for histopathological detection（HE,Masson,and oil red O staining）,oxidative stress indexes and HYP concentration detection,RT-q PCR,and western blot detection.The ileum was collected for HE,RT-q PCR,and western blot detection.The results showed that:（1）Biochemical results showed that Res significantly reduced the serum ALT and AST activities induced by inorganic mercury exposure.（2）Res significantly alleviated the increase of BAC induced by inorganic mercury exposure,indicating that Res significantly alleviated the endogenous ethanol metabolism disorder induced by inorganic mercury in mice.（3）Res significantly reduced the increase in serum LPS concentration induced by inorganic mercury,indicating that Res significantly reduced the damage to the intestinal barrier in mice.（4）The results of liver pathological changes showed that Res significantly reduced the liver damage induced by different doses of inorganic mercury exposure.（5）Res significantly reduced the increase in MDA level and GSH concentration and SOD activity induced by inorganic mercury in mice,indicating that Res significantly inhibited oxidative stress induced by inorganic mercury exposure.（6）Res significantly reduced the increase in liver HYP levels induced by inorganic mercury exposure.（7）RT-q PCR and western blot results of ileal tissue showed that Res significantly reduced the m RNA expressions and protein levels（P<0.05）induced by inorganic mercury exposure,indicating that Res significantly reduced intestinal barrier damage in inorganic mercury exposed mice.（8）Res significantly reduced the m RNA expressions and protein levels of Hmgcr,Cyp7a1 and Cyp39a1（P<0.05）,indicating that Res significantly reduced the abnormalities of liver cholesterol and bile acid metabolism induced by inorganic mercury exposure.（9）Res significantly reduced the reduction of m RNA expressions and protein levels（P<0.05）of inorganic mercury-induced PGC-1α,G6pc,Pdk4 and silent information regulator 1（Sirt1）,indicating that Res alleviated inorganic mercury-induced glucose metabolism abnormalities through activation of the Sirt1/PGC-1αsignaling pathway.（10）Res significantly reduced m RNA expressions and protein levels of nuclear factor erythroid 2-related factor 2（Nrf2）,quinone oxidoreductase 1,and heme oxygenase-1 induced by inorganic mercury,and elevated the Cyp2e1 m RNA expression and protein level（P<0.05）,indicating that Res alleviated inorganic mercury-induced oxidative stress through activation of the Nrf2 pathway.（11）Res significantly reduced the increased levels of m RNA and protein（P<0.05）of inorganic mercury-induced SAA1,tumor necrosisαfactorαand nuclear factor kappa-B,indicating that Res reduced inorganic mercury-induced inflammation.（12）Res administration significantly reduced the changes the levels of Cdh1,Rbp1,Cdkn1a,and Ccnd1（P<0.05）induced by inorganic mercury exposure,indicating that Res significantly reduced inorganic mercury-induced cell cycle abnormalities.（13）Res significantly reduced the levels of transformation growth factorβ1（TGF-β1）,SMAD family member 3,collagen type I alpha 1 andα-smooth muscleαactin induced by inorganic mercury exposure（P<0.05）,indicate that Res alleviates inorganic mercury-induced liver fibrosis through inhibition of the TGF-β1/Smad3 signaling pathway.Part 3 Trial:Bifidobacterium intervention in inorganic mercury poisoning test in mice.Twenty-eight Kunming mice were randomly divided into 4 groups of 7 in each group.The control group drank water normally.The Hg group was given with drinking water containing 100 mg/L of Hg Cl₂.The Hg+BL group mice were given with drinking water containing 100 mg/L of Hg Cl₂,and gavage with BL 5×10⁹ cfu.The BL group was given normal drinking water,and gavage BL 5×10⁹ cfu.Mice eat freely.The trial period was 24 weeks.Mouse serum was collected for liver function biochemical indexes,BAC and LPS concentrations.Liver tissues were collected for oxidative stress indexes and HYP concentration detection,RT-q PCR,and western blot detection.The ileum was collected for RT-q PCR and western blot detection.The results showed that:（1）BL significantly reduced liver injury,endogenous ethanol metabolism disorder,and intestinal barrier damage in mice induced by inorganic mercury exposure.（2）BL alleviates liver cholesterol and bile acid metabolism,glucose metabolism and oxidative stress induced by inorganic mercury through activation of the Sirt1/PGC-1αsignaling pathway.（3）BL alleviates inorganic mercury-induced liver fibrosis through inhibition of the TGF-β1/Smad3 signaling pathway.In summary,long-term and low-dose inorganic mercury exposure induced liver fibrosis in mice through the microbial-gut-hepatic axis,and the main mechanisms included endogenous ethanol metabolism disorders,abnormal cholesterol and bile acid metabolism,abnormal glucose metabolism,oxidative stress,abnormal cell cycle,and inflammation.Res alleviates inorganic mercury-induced liver fibrosis by increasing the relative abundance of Bifidobacterium through the activation of Sirt1/PGC-1αsignaling pathway and balancing the intestinal flora.