| BackgrondModern people living standard is improving, but people’s dietary habits andlifestyle created serious imbalances, a growing number of people is now living in anunhealthy lifestyle, this important risk factor for noncommunicable diseases incidenceof obesity, has severely threatening human health. Obesity means a certain degree ofobvious overweight and thick fat layer, is the body fat, especially a state cause bytriglycerides accumulation, the increased number of fat cells and fat is the mostimportant reason to lead to obesity. Adipose tissue is obesity target organ, one of itsmain function is the regulation of body weight, and triglyceride is the molecular basisof obesity, fat tissue can secrete a lot of acrive substances called adipocytokines whichplay a important role in maintaining energy and stable cardiovascular internalenvironment, glucose and lipid metabolism, immune response, etc, closely related tometabolic syndrome caused by insulin resistance such as obesity,diabetes mellitus andcardiovascular disease.Adipose tissue including adipocyte, preadipocyte,MEC and ECM.The number ofadipose tissue depends on the number of fat cells and volume, the traditional methodof reducing weight is mainly reducing the intake of energy and strengthening theexercise, but it didn’t reduce the number of fat cells, mostly would rebound, and it’slittle effect.Therefore, how to reduce the number of fat cells become our focus ofattention.because the fission of preadipocyte caused to the increasing number ofadipocyte,while apoptotic of preadipocyte and adipocyte brings to the reduce of fattissue,so restrain preadipocyte turns to adipocyte or promote apoptotic ofpreadipocyte and adipocyte could hit the mark of reducing adipose tissue.the same time,reducing the over-dose cumulation of adipose tissue leads to insulin resistance isof great significance of preventive treatment of metabolic syndrome such as fat,glycuresis and CVD.Traditional Chinese medicine long pepper has an function of dispelling cold,stopping cold. Clinical used in the treatment of epigastric pain, vomiting,diarrhea,umbilical abdominal pain, toothache, headache, etc., Mongolian medicine use it in thetreatment of coronary heart disease. Piperine extracted from the long pepper hassignificant hypolipidemic activity. Previous studies have found that alcohol extractedfrom long pepper has the function of improving insulin resistance.This topic on thebasis of previous research, continue to study main active ingredients of Piperine(Piperine, PIP) of3T3-L1the interference of fat cells, explore the Piperine regulatingmechanism of fat cells.Purposes1. Research on the normal3T3-L1and discuss PIP-the influence of L1before fat cellgrowth and function.2. Research on the normal3T3-L1and discuss PIP before fat cells growth inhibitioneffect is through the apoptosis pathway. For the next step in-depth study PIPimprovement of cell and molecular mechanism of insulin resistance to provideexperiment basis.Method1. Determined by MTT method to detect the cell inhibition rate under differentconcentrations of PIPLiving cells mitochondrial succinate dehydrogenase in can make the exogenousdetermined by MTT reduction for water insoluble violet crystal armor Formazan anddeposition in cells, the cells die without this feature. According to the measuredabsorbance value (OD value) watch PIP after different concentration and differenttime of3T3-Ll before fat cell proliferation, the influence of the amount to judge theliving cells, the OD value, the greater the cell activity. Fitting curve can be obtained by computer PIP on3T3-L1of IC50, lay the foundation for the following experiments.2. The single cell gel electrophoresis method to detect apoptosisSingle cell gel electrophoresis method contains a lot of DNA fragments can bedetected the apoptosis of cells. Nucleated cell DNA molecular weight is large, in theDNA are attached to the nuclear matrix superhelix structure, using agarose gel cellembedding on the slide, when all kinds of endogenous and exogenous DNA damagefactors induced cell DNA chain rupture, its superhelix structure damage, in the role ofcell lysis solution, the cell membrane and nuclear membrane, membrane structure isdamaged, protein, RNA in cells and other components are spread throughout the celllysis solution. By fluorescence microscopy, can distinguish between normal cells andapoptosis cells in morphology.3. The method of flow cytometry to detect cell apoptosis related indicatorsThrough the cell cycle detection, phosphatidylserine valgus and mitochondrialmembrane potential detection analysis, dead cells and apoptotic cells can bequantitatively distinguish, observation of PIP’s impact on fat cells apoptosis.Conclusion1. The cultivation of the fat cells before3T3-L1:By cell morphology compare the results of identification experiments create3T3-L1cells as normal before fat cells, to lay the foundation for the next experiment.2. Piperine’s effect on3T3-L1preadipocyte vitalityDifferent concentrations of piperine of3T3-L1preadipocyte vitality hasinhibitory effect, and the quantity of a certain effect. Piperine effect after24h, theconcentration of more than10μ M piperine influence on the absorbance value havesignificant differences, fitting curve by the software, get cell growth inhibition rate ofIC50piperine concentration is108.7μM, indicated that high concentration of piperine3T3-L1preadipocyte have strong inhibition effect; Piperine role after48h, of3T3-L1 preadipocyte had the inhibitory effect of strong, bigger to cell damage.3. Piperine’s effect on3T3-L1cell apoptosisUnder the action of different concentrations of piperine of3T3-L1cells appearearly apoptosis phenomenon, and a certain concentration-response relationship:piperine effect after24h,40μ M and80μ M group on the number of apoptotic cellscompared with control group with significant difference (P <0.05); Piperine role after48h,20μ M,40μ Mand80μ M group, the apoptosis of cells was compared with thecontrol group had significant difference (P <0.001). Prompt piperine3T3-L1beforefat cells can be induced to apoptosis.4. Discussion of Pepper cut of3T3-L1preadipocyte apoptosis mechanismUnder the action of piperine,3T3-L1before fat cells in the changes of cell cycle,especially40μ M group and80μ M cell state is abnormal, may affect the key controlpoints of cells in S phase, blocking the synthesis and replication of DNA, to stop theprocess of cell cycle.Mitochondrial membrane potential change is an early event in cell apoptosis, inunder the action of piperine,3T3-L1before fat cells decrease of mitochondrialmembrane potential was dose dependent,20μ M,40μ M group and80μ M groupcompared with the control group had significant difference (P <0.05).ConclusionPiperine has an function on restraining multiplication and inducing apoptosis ofthe mouses’s3T3-L1preadipocyte and there is a certain amount of activityrelationship. Mechanism may be piperine by activating mitochondrial pathway, andblocking DNA synthesis and replication, the S phase of cell cycle retardation andinhibiting proliferation of3T3L1before fat cells, and can promote cell apoptosis.Due to the high concentration of piperine solution has strong cytotoxicity, so we needfurther research that it’s effects on metabolic diseases and mechanism under lowconcentration. |
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