| Cadmium is a common pollutant in the environment,and microplastics have become one of the new pollutants in recent years.Both cadmium and microplastics can enter the body through various routes and cause damage to various organs.Microplastics can also adsorb cadmium in the surrounding environment and exert a toxic effect in combination.The liver is one of the main target organs of cadmium and microplastics.Studies have shown that both cadmium and microplastics can destroy the normal morphology of liver cells,cause oxidative damage,and induce lipid accumulation in the liver,increased autophagy levels,and autophagy flow blockade.CPT1 is a key rate-limiting enzyme for the β oxidation of fatty acids,and studies have found that activating CPT1 can increase the rate of β oxidation and slow lipid accumulation in the liver.In this experiment,C57BL/6 mice and AML 12 cells of normal hepatocyte line of mice were used as the research objects,and mice and cells were treated with polystyrene microplastics and cadmium chloride alone or in combination,and through in vivo experiments and in vitro experiments,cell molecular biology and other related methods were used to explore whether microplastics could exacerbate cadmium-induced hepatocyte damage,lipid accumulation and autophagy flow blockade,and activate CPT1 to alleviate hepatocyte fat accumulation and further alleviate autophagy block caused by abnormal lipid accumulation.It provides a new theoretical basis for the targeted prevention and treatment of liver injury caused by cadmium and microplastics.1.Exposure to microplastics and cadmium causes Liver damageSPF-grade 6-week-old male C57BL/6J mice were pre-rearing one week and randomly divided into six groups(n=8 each)。The control group provided DDW,the Cd group provided 50 mg/L CdC12 solution,the 5 μm PS-MPs group provided 10 mg/L 5 μm PS-MPs suspension,the Cd+5 μm PS-MPs group provided 50 mg/L CdCl2+10 mg/L 5 μm suspension,the 0.5 μm PS-MPs group provided 10 mg/L 0..5 μm PS-MPs suspension,and the Cd+0.5 μm PS-MPs group provided 50 mg/L CdCl2+10 mg/L 0.5 μm suspension.Experiments were conducted after three months of rearing.AML12 cells were divided into control group,Cd group,5 μm PS-MPs group,Cd+5 μm PS-MPs group,Cd concentration was 5 μmol/L,PS-MPs concentration was 100 μg/mL.The results showed that both Cd and PS-MPs could enter the liver and hepatocytes and accumulate.Cd and PS-MPs significantly reduced the body weight and liver coefficients of mice(P<0.01),and the Cd+5 μm PS-MPs group further reduced the liver coefficients(P<0.01).HE staining results and TEM results showed that Cd and PS-MPs exposure destroyed the morphological structure of the liver,and the addition of PS-MPs exacerbated the damage of Cd to the liver.Compared with the control group,the content of trace elements Mn and Zn in the treatment group was significantly or very significantly increased(P<0.05 或P<0.01),the Cu content was significantly or very significantly reduced(P<0.05或 P<0.01),the content of MDA was significantly increased(P<0.05 或P<0.01),and SOD,T-AOC and GSH were significantly reduced(P<0.05 或 P<0.01),indicating that the exposure of Cd and PS-MPs caused oxidative stress in the liver,while PS-MPs exacerbated the degree of Cd-induced oxidative stress in the liver.The cell morphology was observed in brightfield microscopy,and the Cell Real-Time Analysis System(RTCA)detected the cell index,and the results showed that Cd and PS-MPs alone or in combination exposure would have an impact on cell morphology,but PS-MPs alone had little effect on the cell index.In summary,PS-MPs can enter and accumulate in mouse liver and AML12 cells;The exposure of Cd and PS-MPs alone or in combination significantly reduced the weight and liver coefficients of mice,and the exposure of PS-MPs intensified the effect of Cd on liver coefficients.PS-MPs exposure exacerbates Cd-induced liver damage and oxidative stress.2.Exposure to microplastics and cadmium causes hepatic lipid accumulation and autophagyThe C57BL/6 mouse and the AML12 cells of the mouse were treated in the same way as in the previous section to explore lipid accumulation and autophagy in the liver caused by exposure to microplastics and cadmium.HE staining and TEM results showed a large accumulation of lipid droplets in the liver.Compared with the control group,the TG content of the treatment group was significantly increased(P<0.05或P<0.01),the contents of T-CHO,LDL-C and VLDL were significantly or very significantly reduced(P<0.05 或P<0.01),the content of Cr elements was significantly increased(P<0.05或P<0.01),compared with the Cd group,the contents of T-CHO,TG,HDL-C,Cr in the Cd+5 μm PS-MPs group were significantly increased(P<0.05 或 P<0.01),and the contents of T-CHO,HDL-C and Cr elements in the Cd+0.5 μm PS-MPs group were significantly increased(P<0.05或P<0.01).The results of western blotting showed that compared with the control group,the ACC content in the treatment group was significantly increased(P<0.05或P<0.01),and the expression of PPARa,CPT1,apoB and MTP decreased significantly or very significantly(P<0.01).Compared with the Cd group,the ACC was significantly increased in the Cd+5 μm PS-MPs group(P<0.05),the expression of PPAR-a,CPT1,apoB,and MTP decreased significantly(P<0.05 或P<0.01),and the PPAR-α in the Cd+0.5 μm PS-MPs group decreased significantly(P<0.01).For AML 12 cells,oil red O staining and lipid droplet fluorescence staining observed a large accumulation of lipid droplets within the cells of the treated group.Compared with the control group,the contents of T-CHO and TG in the treatment group were significantly increased(P<0.05或P<0.01),and compared with the Cd group,the contents of T-CHO and TG in the Cd+5 μm PS-MPs group were further increased(P<0.05 或P<0.01).The results of western blotting showed that the expression of ACC was very significantly increased(P<0.01),and PPAR-α and CPT1 were significantly reduced in the treatment group(P<0.05 或P<0.01).Compared with the Cd group,the expression of ACC was significantly increased in the Cd+5μm PS-MPs group(P<0.01),and PPAR-α and CPT1 were significantly reduced(P<0.05或P<0.01).The above results showed that PS-MPs and Cd exposure caused lipid metabolism disorders and abnormal lipid accumulation in mouse liver and AML 12 cells,and PS-MPs intensified the lipid accumulation of Cd induced liver and hepatocytes.For the study of autophagy levels,TEM observed a large number of autophagic bodies in the liver tissue of the treatment group,and the western blotting results showed that the expression of ATG7,Beclinl,P62 and LC3Ⅱ in the treatment group were significantly increased compared with the control group(P<0.05 或P<0.01).Compared with the Cd group,the expression of ATG7,Beclinl,P62 and LC3Ⅱ in the Cd+5 μm PS-MPs group was significantly increased(P<0.05 或P<0.01),and Beclin1 and P62 in the Cd+0.5 μm PS-MPs group were significantly increased(P<0.05 或P<0.01).On AML12 cells,western blotting results showed that the expression of ATG7,P62 and LC3II in the treatment group was significantly higher than in the control group(P<0.05),and the expression of ATG7 and LC3Ⅱ in the Cd+5 μm PS-MPs group was significantly increased compared with the Cd group(P<0.05).RFP-GFP-LC3 tandem fluorescent protein expression assay observed that PS-MPs and Cd exposure led to autophagic flow blockade.PS-MPs exacerbate Cd exposure-induced autophagy flow arrest.The above results showed that PS-MPs and Cd exposure increased the autophagy levels of mouse liver and AML12 cells,and PS-MPs exacerbated the increase in autophagy levels and autophagy flow blockade of Cd-induced liver and liver cells.3.Regulation of CPT1 alleviates lipid accumulation and autophagy flow arrest induced by microplastic and cadmium exposureTaking the AML12 cells as the research object,the four treatment groups were:control group,100μmol/L Baicalin group,5μmol/L Cd+10 μg/mL 5 μm PS-MPs group,and 5μmol/L Cd+10 μg/mL 5 μm PS-MPs+100 μmol Baicalin group,to explore whether activating CPT1 could alleviate lipid accumulation and autophagy flow blockade induced by microplastic and cadmium exposure.The results of lipid droplet fluorescence staining showed that there was a large accumulation of lipid droplets in the Cd+5 μm PS-MPs group,while the content of lipid droplets in the Baicalin+Cd+5 μm PS-MPs group was reduced.Compared with the control group,the T-CHO and TG levels increased very significantly in the Cd+5 μm PS-MPs group(P<0.01),while the Baicalin+Cd+5 μm PS-MPs group decreased significantly(P<0.05 或 P<0.01).The western blotting results showed that compared with the control group,the expression of ACC and PPARα in the Cd+5 μm PS-MPs group increased significantly(P<0.05).After activating CPT1,compared with the infected group,the expression of ACC decreased significantly,and the expression of PPARa increased significantly(P<0.05).The results of RFP-GFP-LC3 tandem fluorescent protein showed that compared with the control group,the yellow aggregation points in the Cd+5 μm PS-MPs group increased significantly(P<0.05),and the number and brightness of yellow aggregation points were reduced to a certain extent compared with the infected group after activating CPT1.The western blotting results showed that the expression of ATG7,P62 and LC3Ⅱ in the Cd+5μm PS-MPs group was significantly higher than that in the control group(P<0.05),and after activating CPT1,the expression of ATG7,P62 and LC3II was significantly reduced compared with the infected group(P<0.05).In summary,after activating CPT1,the lipid droplets in hepatocytes were significantly reduced,the level of autophagy was reduced,and the autophagy flow was smooth.It was indicated that activating CPT1 could effectively alleviate PS-MPs and Cd-induced lipid accumulation in hepatocytes,thereby alleviating autophagy flow blockade caused by abnormal lipid accumulation.In conclusion,microplastics and cadmium can cause lipid accumulation and autophagy in the liver and block autophagy flow,and the combined exposure further aggravates liver toxic injury.Activation of CPT1 can alleviate lipid accumulation in hepatocytes induced by cadmium and microplastics,reduce the level of autophagy,and alleviate the block of autophagic flow. |
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