Study On The Effects And Mechanism Of Ascaris Body Cavity Fluid On Proliferation And Differentiation Of 3T3-L1 Preadipocytes

This study explores the influence of Ascaris body cavity fluid(ABF)on the proliferation, differentiation and apoptosis of mouse 3T3-L1 preadipocytes, to reveal the mechanism of action of ABF on the body lipid metabolism. Test methods: Collected Ascaris body cavity fluid, then diluted the initial concentration 3200μg/mL of ABF to 3000μg/mL、2000μg/mL、1000μg/mL、800μg/mL、600μg/mL、400μg/mL、200μg/mL、100μg/mL、50μg/mL and 10μg/mL as the test material. Establish the 3T3-L1 cells as the experimental model, respectively set the cell proliferation group, cell apoptosis group, the differentiation group and the PPARγmRNA expression group. Cell proliferation group: cells in the logarithmic phase were counted and inoculated into 96-well cultivation palte exactly. ABF with initial concentration of 3200μg/mL was diluted mutltiproportionly into 3000μg/mL, 2000μg/mL, 1000μg/mL, 800μg/mL, 600μg/mL, 400μg/mL, 200μg/mL, 100μg/mL, 50μg/mL and 10μg/mL. The method of MTT was used to test the vitality of the cells. Observe the influence of different concentrations of ABF to 3T3-L1 proliferation in different time periods. Cell apoptosis group: set 1000μg/mL ABF group and control group, Cells in each group were collected after 36h, the cell apoptotic rate was detected by flow cytometry. The differentiation group: induce the logarithmic growth phase cells to differentiate, respectively set 3000μg/mL, 2000μg/mL, 1000μg/mL, 500μg/mL, 100μg/mL, 50μg/mL and 10μg/mL ABF group, add the corresponding concentrations of the ABF to differentiation process, the oil red O staining was used after differentiation, then measured the absorbance at 510nm wavelength. The PPARγmRNA expression test group: set 1000μg/mL, 100μg/mL ABF group and control group, the cell RNA were extracted by RT-PCR method for the detection of PPARγand GAPDH mRNA expression after the end of the differentiation.The results as follow:1. Cell proliferation results: high concentrations of ABF can significantly inhibit 3T3-L1 cell proliferation, low concentrations of ABF (<10μg/mL) can stimulate the proliferation of 3T3-L1 cells. At the same concentration, the ABF(> 10μg/mL) inhibition increased with increasing time.2. Cell apoptosis results: adding 1000μg/mL ABF during the differentiation process, the rate of apoptosis increase from 15.99% to 25.87%, indicate that ABF can significantly increase the rate of apoptosis.3. Cell differentiation staining results: high concentrations of ABF(2000μg/mL、1000μg/mL、500μg/mL) can significantly reduce the intracellular fat (P<0.01), the fat of intracellular production was 26.5%, 55.5% and 81.5% compared with the control group. The difference of intracellular fat between low concentrations of ABF(100μg/mL, 50μg/mL, 10μg/mL)group and control group was not obvious, even higher than control group, but the difference was not statistically significant (P> 0.05).4. PPARγmRNA expression results: PPARγmRNA expression was significantly declined by adding 1000μg/mL ABF (P<0.01) during preadipocyte 3T3-L1 differentiation process, was57.6% as the control group. The PPARγmRNA expression level in 10μg/mL ABF group was not significant compared with the control group (P> 0.05).Conclusion:1. The intervention of ABF on lipid metabolism may be closely related with the inhibition to the proliferation of 3T3-L1 cells.2. The intervention of ABF on lipid metabolism may be closely related with the promotion to the apoptosis of 3T3-L1 cells.3. The inhibition of ABF to 3T3-L1 preadipocyte transformed into mature fat cells may be an important mechanism to interfere with lipid metabolism.4. The main mechanism of ABF inhibition of preadipocyte 3T3-L1 into mature fat cells is to reduce the expression of PPARγmRNA.ABF significantly reduce the amount of lipogenesis by a number of ways, it has a significant role in regulating lipid metabolism.