Protective Effect Of Sodium Humic Acid On Intestinal And Liver Injury Induced By Polystyrene Microplastics In Mice

Microplastics(MPs)are plastic fragments that are widely distributed in the environment and accumulated in organisms.A large number of studies have shown that MPs accumulates in the brain,heart,liver,spleen,lung,kidney,intestinal tract,uterus and testis of aquatic organisms and mammals through respiratory,digestive and skin contact,resulting in oxidative stress,inflammatory reaction,immune injury,intestinal microflora imbalance and energy metabolism disorder.However,at present,most studies focus on the toxicological effects of polystyrene microplastics(PS MPs),and there are few studies on how to alleviate the harm caused by MPs.It has been found that humate acid can alleviate the head kidney damage caused by European perch exposed to polystyrene nanoplastics and has protective anti-inflammatory effects.Sodium humate(HA)used in this study is a kind of humic acids,which has antibacterial and anti-inflammatory,hemostatic and diarrhea,improve production performance and enhance immune function,and has a protective effect on the intestinal tract.The purpose of this study is to explore the protective effect of HA on intestinal and liver injury induced by PS MPs in mice,and to further explore its protective mechanism.The study randomly divided 30 male Kunming mice into 5 groups,with 6 in each group,namely the blank control group(CON)and the MPs group(1000 μg/L)and different doses of HA(0.2%,0.4%,0.6%)+ MPs groups.The experimental period is 6 weeks,during which the mice freely consume and drink water and record changes in body weight.After the experiment,collect mouse serum,liver,colon,and cecal contents,record the weight of mouse liver,and conduct subsequent experiments.Colonic and liver tissues were stained with HE and colonic tissues were stained with AB-PAS.The content of Mucin-2 in colon was detected by immunofluorescence technique and the expression of ZO-1,Occludin and Claudin-1 in tight junction of colon was detected by immunohistochemistry.ELISA kit was used to detect LPS,DAO and s Ig A in serum of mice.Biochemical kits were used to detect the contents of ALT,AST,HDL-C,T-CHO,TG and TBA in serum as well as T-AOC,GSH and MDA in colon and liver.The levels of m RNA and protein expression of colonic tight junction(ZO-1,Occludin and Claudin-1),adhesion junction(E-cadherin,β-catenin),epithelial proliferation related factors(EGFR,PCNA and TGF-β1),inflammatory factors IL-1β,IL-6,IL-10,TNF-α in colon and liver and TLR4/NF-κB pathway related factors in liver tissue were detected by real-time fluorescence quantitative PCR and Westernblot.The V3-V4 region of 16 S r DNA gene was sequenced to study the changes of cecal microflora in CON group,MPs group and MPs-M-HA mice.The test results are as follows:(1)MPs exposure could damage colonic epithelial cells,destroy crypt structure,decrease the number of goblet cells and mucin Mucin-2 secretion,increase intestinal permeability,increase the contents of LPS,DAO and D-Lac in serum,decrease the content of s Ig A in colonic tissue,and destroy intestinal structure and function.HA intervention can increase the number of colonic goblet cells,promote mucin secretion,prevent the passage of pathogenic microorganisms,and then improve intestinal barrier function.(2)MPs exposure significantly decreased the m RNA and protein expression of ZO-1,Occludin,Claudin-1 and adhesion junction E-cadherin and β-catenin in colonic tight junction,and destroyed intestinal tight junction and adhesive junction.HA intervention can enhance intestinal tight junction and adhesion junction,promote intestinal epithelial cell proliferation related factors EGFR,PCNA and TGF-β1 m RNA and protein expression,and enhance intestinal barrier function.(3)Compared with the blank control group,MPs exposure could lead to the increase of liver index,hepatomegaly,hepatocyte vacuolation and eosinophil,suggesting inflammatory reaction in the liver,the content of ALT and AST increased,the content of TBA increased,while the content of T-CHO,TG and HDL-C decreased,indicating abnormal liver function and disorder of lipid metabolism.After intervention with HA,the structure and function of liver were significantly improved.(4)MPs exposure could decrease the activity of T-AOC,decrease the content of GSH and increase the content of MDA in colon and liver of mice.HA intervention could increase the levels of T-AOC and GSH and decrease the content of MDA,indicating that HA has the effect of antioxidation.Compared with CON group,the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α in MPs group increased significantly,while the anti-inflammatory factor IL-10 decreased significantly.HA intervention could reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors,indicating that HA can regulate inflammatory factors and has anti-inflammatory effects.(5)Compared with the control group,MPs exposure resulted in increased intestinal permeability.Intestinal bacteria and their metabolites LPS flowed into the liver through the damaged intestinal barrier and upregulated the expression of TLR4/ NF-κB signaling molecules TLR4,My D88,NF-κB,p-NF-κB,IκBα and p-IκBα,and the m RNA expression of TLR4,IRAK4,TRAF6,My D88,NF-κB and IκBα.HA intervention inhibited the activation of pathway-related factors.And then inhibit the release of inflammatory factors,play a role in protecting the intestinal tract and liver.(6)The results of intestinal flora sequencing showed that MPs exposure significantly increased the abundance of Saccharibacteria,while Saccharibacteria played a proinflammatory role in gastrointestinal diseases and significantly decreased the abundance of Lactobacillus.HA intervention can increase the abundance of intestinal beneficial bacteria such as Lactobacillus,Marvinbryantia,Sutterella and so on.MPs exposure can lead to the disorder of intestinal flora,change the diversity of microbiota,increase the abundance of harmful bacteria,further destroy intestinal barrier,aggravate inflammatory reaction and intestinal injury.HA can increase the abundance of intestinal beneficial bacteria,regulate intestinal flora homeostasis,and then inhibit liver inflammation mediated by TLR4/ NF-κB pathway induced by LPS.To sum up,HA can improve the structure and function of the intestine and liver,enhance the tight connection of the intestinal barrier,inhibit oxidative stress and inflammatory reactions in the intestine and liver,regulate the abundance of intestinal microbiota,and prevent LPS from entering the intestinal liver circulation to activate TLR4/NF-κB signaling pathway effectively alleviates intestinal and liver damage caused by MPs.