Integration of transcriptome, gut microbiota, and physiology reveals toxic responses of the red claw crayfish (Cherax quadricarinatus) to imidacloprid.

Imidacloprid enters the water environment through rainfall and causes harm to aquatic crustaceans. However, the potential chronic toxicity mechanism of imidacloprid in crayfish has not been comprehensively studied. In this study, red claw crayfish (Cherax quadricarinatus) were exposed to 11.76, 35.27, or 88.17 µg/L imidacloprid for 30 days, and changes in the physiology and biochemistry, gut microbiota, and transcriptome of C. quadricarinatus and the interaction between imidacloprid, gut microbiota, and genes were studied. Imidacloprid induced oxidative stress and decreased growth performance in crayfish. Imidacloprid exposure caused hepatopancreas damage and decreased serum immune enzyme activity. Hepatopancreatic and plasma acetylcholine decreased significantly in the 88.17 µg/L group. Imidacloprid reduced the diversity of the intestinal flora, increased the abundance of harmful flora, and disrupted the microbiota function. Transcriptomic analysis showed that the number of up-and-down-regulated differentially expressed genes (DEGs) increased significantly with increasing concentrations of imidacloprid. DEG enrichment analyses indicated that imidacloprid inhibits neurotransmitter transduction and immune responses and disrupts energy metabolic processes. Crayfish could alleviate imidacloprid stress by regulating antioxidant and detoxification-related genes. A high correlation was revealed between GST, HSPA1s, and HSP90 and the composition of gut microorganisms in crayfish under imidacloprid stress. This study highlights the negative effects and provides detailed sequencing data from transcriptome and gut microbiota to enhance our understanding of the molecular toxicity of imidacloprid in crustaceans.

[Effects of ursolic acid on c-Cbl-associated protein expression in 3T3-L1 adipocytes with insulin resistance].

OBJECTIVE: To observe the effects of ursolic acid (UA) on insulin resistance and cell differentiation in 3T3-L1 adipocytes and to explore the mechanisms. METHODS: 3T3-L1 adipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with glucose (25 mmol/L) and insulin (10(-6) mol/L) to induce insulin resistance. After culture, glucose consumption of the adipocytes was detected by glucose oxidase method and glucose uptake was detected by using tritium-marked glucose. Drug concentration for following test was determined through detecting the effects of different concentrations of UA on the activity of 3T3-L1 adipocytes with insulin resistance by methyl thiazolyl tetrazolium (MTT) staining. 3T3-L1 adipocytes with insulin resistance were cultured with DMEM, rosiglitazone, and low- and high-dose UA, and then, glucose uptake and differentiation of 3T3-L1 adipocytes were detected. Finally, real-time fluorescence quantitative polymerase chain reaction and Western blot methods were used to detect the effects of UA on expressions of adipocyte lipid binding protein (aP2), c-Cbl-associated protein (CAP) and matrix metalloproteinase-1 (MMP-1) in 3T3-L1 cells with insulin resistance. RESULTS: After dealing with high glucose/hyperinsulin for 24 h, insulin resistance was induced successfully in the 3T3-L1 adipocytes. The concentrations of UA were defined to be 4 to 20 µmol/L. Compared with the model group, the glucose uptake was significantly increased in the rosiglitazone group and groups treated with low- and high-dose UA (P<0.01). The differentiation levels of 3T3-L1 adipocytes in the UA groups were lower than those in the control group and the rosiglitazone group. Effects of UA on the expressions of aP2 and MMP-1 were not obvious, but UA could up-regulate expression of CAP both in mRNA and protein levels (P<0.01). CONCLUSION: Low- and high-dose UA can improve the glycometabolism and differentiation of 3T3-L1 adipocytes with insulin resistance by up-regulating the expression of CAP.

[Effects of ursolic acid in ameliorating insulin resistance in liver of KKAy mice via peroxisome proliferator-activated receptors α and γ].

OBJECTIVE: To explore the effects and mechanism of ursolic acid in improving hepatic insulin resistance in KKAy mice with spontaneous type 2 diabetes. METHODS: Thirty-five KKAy mice were divided into five groups according to the randomized block design, namely, control, rosiglitazone, fenofibrate, and high- and low-dose ursolic acid groups with seven mice in each group. C57BL/6J mice were used as the normal control group. At the end of the 4th week, free fatty acid (FFA), tumor necrosis factor-α (TNF-α) and adiponectin contents in serum were detected by enzyme-linked immunosorbent assay; the protein expressions of phosphoenolpyruvate carboxykinase (PEPCK), insulin receptor substrate-2 (IRS-2) and glucose transport factor-2 (GLUT-2) were detected by Western blot method; the mRNA expressions of PEPCK, IRS-2 and GLUT-2 were detected by real-time polymerase chain reaction; the expressions of peroxisome proliferator-activated receptor α (PPARα) and peroxisome proliferator-activated receptor γ (PPARγ) in liver tissue were detected by immunohistochemical method. RESULTS: After four weeks of intervention, the contents of FFA, TNF-α and adiponectin in serum of the high-dose ursolic acid group had changed, showing statistically significant difference compared to those of the control group (P<0.01); high dose of ursolic acid had depressant effect on the expressions of PEPCK protein and PEPCK mRNA (P<0.01); low dose of ursolic acid depressed the expression of PEPCK mRNA and induced phosphorylation of IRS-2 in the liver (P<0.05); both high and low dose of ursolic acid improved the expression of PPARα in the liver (P<0.01). CONCLUSION: The effects of ursolic acid in improving hepatic insulin resistance in KKAy mice with spontaneous type 2 diabetes may be closely related to affecting the contents of FFA, TNF-α and adiponectin, improving the expression of PPARα protein, regulating transcription of PEPCK protein and inducing phosphorylation of IRS-2.