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Pentamethylquercetin Browns the White Adipocytes and the Study of Adult Mouse Cardiac c-Kit~+ Cells

Author: HanYi
Tutor: JinManWen
School: Huazhong University of Science and Technology
Course: Pharmacology
Keywords: pentamethylquercetin UCP-1 white adipose tissue brown adipose tissue high fat diet pharmacokinetic ion channels cardiac c-kit~+ cells ICLvol
CLC: R285.5
Type: PhD thesis
Year: 2011
Downloads: 50
Quote: 0
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Abstract


Object:To study the effects of PMQ on 3T3-L1 adipocytes. Method:MTT, Oil red O stain, Janus Green B stain, the commercial kits of Glu, TG, and Glycerol, RT-PCR, Western blot and Realtime PCR were used to evaluate the effects of PMQ on the proliferation, differentiation, glucose consumption, cellular lipid metabolism, Mitochondria biogenesis, and the expression of related brown genes in 3T3-L1 adipocytes. Results:PMQ had no effect on the proliferation of 3T3-L1 preadipocytes. However, PMQ treatment upregulated the consumption of glucose, inhabited the accumulation of cellular TG, induced the biogenesis of Mitochondria and expression of UCP-1, PGC-1α, C/EBPα, PRDM16, PPARγ, PPARα. Conclusion: PMQ can brown 3T3-L1 adipocytes, and affect on the early stage of adipocyte differentiation.Object:To study the effects of PMQ on adult Kunming mice. Methods:45 adult Kunming mice were randomized into Vehicle, PMQ 2.5,5,10 mg/kg and RSG 5 mg/kg group. Body weights and food intakes were monitored weekly. After 14 days of PMQ treatment, the animals were sacrificed. Brown adipose tissues were weighed, and the expression of brown genes in WAT was analysed. Results:PMQ treatment for a short time had no impact on body weights, food intakes and BAT weights of adult Kunming mice, however the mRNA levels of UCP-1, PGC-1α, and C/EBPαwere upregulated in WAT.Object:To explore the effects of PMQ on high-fat diet fed C57BL/6 mice. Method:36 C57BL/6 mice were randomized into standard-fat diet (SFD), high-fat diet (HFD) and high-fat with 0.04% PMQ diet (HFD+PMQ) group. Body weights and food intakes were monitored weekly. After 20 weeks, the animals were sacrificed. The signs and the blood biochemistry parameters of animals were analysed. The biogenesis of Mitochondria and expression of UCP-1 protein in WAT were measured by immunohistochemistry. The mRNA levels of related brown genes in WAT were tested by Realtime PCR. Results:Compared to the HFD group, HFD +PMQ group had a leaner waistline, a smaller LEE index, a lower WAT weight, but a higher BAT weight. PMQ also could alleviate hyperglycemia, hypertriglyceridemia, hypercholesterolemia and the size of white adipocytes in HFD mice, induce the biogenesis of mitochondria and upregulate the UCP-1 protein level in WAT. Conclusion:The hyperglycemia, hypertriglyceridemia, hypercholesterolemia in HFD mice was attenuated by PMQ, and PMQ also displayed the ability of browning WAT of HFD mice.1. Establishment of HPLC methodsProtein in serum and tissue homogenate was precipitated and concentrated with methanol. PMQ was detected by HPLC. This method showed a good specialty, recurrence and good sensitivity with fine linearity in the concentration range of 0.1~12μg/ml for plasma. The minimum detective concentration was 0.06μg/ml.2. Plasma concentration-time curve and pharmacokinetic parametersThe concentration of PMQ in plasma was determined at different time points after intravenous administration (i.v.) at the dose of 12 mg/kg. The data was analysed by DAS system. The results indicated that the concentration-time (C-T) curve of 12 mg/kg (i.v.) fitted one-compartment model and there were significant gender differences in some of the parameters.The concentration of PMQ in plasma was determined at different time points after intragastric administration at the dose of 20 mg/kg and 50 mg/kg. The data was analysed by DAS system. The results indicated that the concentration-time(C-T) curve of 20 mg/kg and 50 mg/kg (i.g.) both fitted one-compartment model, bioavailability of PMQ was up to 89% and there were significant gender differences in some of the parameters.Object:Cardiac c-kit+ cells are generally believed to be the major population of stem/progenitor cells in the heart. The present study isolated and identified the adult mouse cardiac c-kit+ cells, and explored the effect of 2 major expressed ion channels on the proliferation of undifferentiated cardiac c-kit+ cells. Method: Immunocytochemistry and RT-PCR were used to identify the type of the cells. MTT and Flow cytometry were used to study the effect of iron channels inhibitors: NPPB/Niflumic acid (for ICl.vol),4-AP/TEA (for IKDR) on cardiac c-kit+cell proliferation. Result:The purity of isolated and cultured cardiac c-kit+ cells was up to 96%. ICl.vol participates in regulating cell proliferation.

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