| Objective:(1)To study the the pathogenesis characteristics "phlegm blockage and qi stagnation" of hyperlipidemia.(2)To observe Citrus changshan-huyou Peel flavonoids components on lipid metabolism and the role of PPARs Signaling pathway in HepG2 cells in vitro,To confirm the lipid-lowing function of citrus changshan-huyou peel and the main medicinal substances of citrus changshan-huyou peel,to provide the experimental basis for exploiting and utilizing.Methods:Mechanism of citrus changshan-huyou peel in treatment of hyperlipidemia was studied in aspects of TCM theory and modern pharmacological experiment with the methods combined with literature analysis and experiments.(1)Literature research:The name of hyperlipidemia and in-depth excavation of "fat" and "cream" were studied through ancient and modern literature.The etiology and pathogenesis of hyperlipidemia were interpreted from the function of viscera and physiological metabolism of qi,blood and body fluid,based on"fat and cream theory",combined with "phlegm theory","blood stasis theory" and "qi stagnation theory".The understanding of the etiology and pathogenesis of hyperlipidemia"phlegm blockage and qi stagnation" were further improved combined with in-depth analysis of "irregular diet","over-fatigue" and "emotional disorders".(2)Experimental study:①Firstly,high cholesterol and triglyceride cell models were constructed with the human hepatoma cell line HepG2,then divided intodivided into Model group and control group of 10,interpreted by citrus changshan-huyou peel flavonoids with different concentrations.Then the IC50 of drug was detected with CCK-8 method and calculated for dose screening.Secondly,the optimal dose of total flavonoids was screened by detecting the influence of citrus changshan-huyou peel flavonoids with different concentrations on the expression of PPARa and PPARy in high cholesterol and triglyceride cell models by Western Blot method.Finally,high cholesterol and triglyceride cell models were interpreted by the optimal dose of total flavonoids,and the levels of cholesterol and triglyceride were detected by Elisa method.Then the lipid-lowering function of citrus changshan-huyou peel flavonoids was defined,and the regulating effect and lipid-lowering mechanism were defined combined with the influence of flavonoids on the expression of PPARa and PPARy detected by Western Blot method.②Citrus changshan-huyou peel were extracted by system solvent,and CP(petroleum ether extract),CE(ethyl acetate extract),CB(n-butanol extract)and CW(aqueous phase)were obtained by the gradient extraction of the concentrate.High cholesterol and high triglycerides models were constructed.There were 12 groups in normal group,model group,high and low concentration and different solvent extracts.Contents of cholesterol,oleic acid in cytoplasm of different concentrations and different solvent extracts were detected.The amplification curve and melting curve of PPARa and PPARγ were drawed by real-time PCR,and the expression of PPARa and PPARy in each group were detected by Western Blot method.The impact of different flavonoids components on high cholesterol and high triglycerides inducing HepG2 cells were statistically analyzed,the lipid-lowering effect of each component was defined,and the main active substance with lipid-lowering effect was screened.Results:(1)Literature research showed that hyperlipidemia belongs to the narrow"visible phlegm" in blood,which belongs to "too much grease ".The main causes of it lie in irregular diet,over-fatigue and emotional disorders.The main pathogenesis are dysfunction of lung,body fluid stagnation,production of phlegm and wet;dysfunction of spleen,dampness retention,phlegm generation;deficiency of kidney qi,dysfunction of on-off and qi transformation,phlegm stagnation;liver qi stagnation,dysfunction of abreaction,body fluid turning to phlegm;deficiency of heart qi and heart yang,phlegm and blood stasis;qi stagnation,phlegm and blood stasis are mixed and affect each other.The key of pathogenesis of hyperlipidemia is qi stagnation,phlegm obstruction,phlegm coagulation and blood stasis,blocking blood circulation,so smoothing qi,eliminating phlegm,activating blood circulation and removing blood stasis should be taken into consideration in treatment.The citrus changshan-huyou peel has its unique advantages in hyperlipidemia prevention and treatment.The effects of smoothing qi and eliminating phlegm are corresponding to the pathogenesis characteristics "phlegm blockage and qi stagnation" of hyperlipidemia.It can inhibit absorption of cholesterol,promote the decomposition of fat,accelerate the enterohepatic circulation,regulate the apolipoprotein,regulate vascular endothelial function,scavenge oxygen free radicals and change blood viscosity and blood rheology,which is suitable for the prevention and treatment of hyperlipemia.(2)Experimental study:①Study on the lipid-lowering effect and mechanism of citrus changshan-huyou peel flavonoidsIn cholesterol model,the cholesterol content showed a very significant difference in middle dose and high dose groups compared with the normal group(P<0.01),while there was no statistically significant difference between low dose groupnd normal group.In oleic acid model,the triglyceride content showed significant difference in low dose group compared with the normal group(P<0.05),which showed a very significant difference in middle dose and high dose groups compared with the normal group(P<0.01).Therefore,the high cholesterol model and high oleic acid model were successfully established,and the latter could be modeled with the medium dose.Effect of citrus changshan-huyou peel flavonoids on HepG2 cells was detected by CCK-8.The results showed that compared with the control group,the proliferation inhibition effect of citrus changshan-huyou peel flavonoids of different concentrations on HepG2 cells,increased gradually with the increase of dose,and there were significant differences(P<0.05),suggesting that the inhibitory effect was concentration dependent.The pharmaceutical IC50 at 48h was 3.1mg/ml,which was used as the reference value,and high,middle and low dose of citrus changshan-huyou peel flavonoids were determined to be 5mg/ml,2.5 mg/ml and 1.25mg/ml.The optimal dose of total flavonoids was screened by the expression of PPARa and PPARy detected by WB.Results showed that among the 4 doses of low(1.25mg/ml),middle(2.50mg/ml),high(5mg/ml)and IC50(3.1mg/ml)of citrus changshan-huyou peel flavonoids in high cholesterol and high oleic acid model,middle and high(5mg/ml)doses were more obvious in cholesterol experiment while low and middledoses were more obvious in triglyceride experiment.The contents of intracellular cholesterol and triglyceride were detected by ELISA method.Results showed that the cholesterol in model group was significantly higher than in normal group(P<0.01),while the cholesterol in lovastatin,flavonoids middle dose and high dose groups were significantly lower than in model group(P<0.01),there were no significant difference among the three.The triglyceride content in oleic acid model groupwas significantly higher than in normal group(P<0.01),while it in the fenofibrate group was significantly lower than in model group(P<0.01),it decreased after treatment of low and middle doses flavonoids but without significant difference(P>0.05).The protein expression of PPARa and PPARγ were detected by WB.The protein transfer with gray scale analysis showed that in high cholesterol model,PPARa and PPARγ expression in model group decreased(P<0.01)than in normal group.PPARa and PPARγ expression in middle and high dose groups increased than in model group,while didn’t change obviously in lovastatin group.In high oleic acid model,PPARα and PPARγ expression in model group decreased(P<0.01)than in normal group.PPARα and PPARy expression increased(P<0.01)in middle dose group and in low dose group than in model group(P<0.01).PPARα expression in fenofibrate group increased(P<0.01),while PPARγ expression didn’t change obviously.②Pharmacodynamic material basis of lipid-lowering effect of citrus changshan-huyou peel from PPARs nuclear receptorThe cholesterol content of different flavonoids components in supernatant was not significantly different between normal group and treated group.The oleic acid content of different flavonoids components in supernatant in CW and CB groups were significantly higher than in model group.The content of cholesterol in cytoplasm in CB group was significantly lower than in other groups,and the content of oleic acid in cytoplasm in CE and CW groups were significantly lower than in other groups.In high cholesterol model,the level of cholesterol was found to be closer to the standard curve in CP and CE cytoplasm,according to cholesterol standard curves and comparison of OD of different flavonoids components at the 550nm.In high triglyceride model,according to oil red O staining,the lipid in model group was more than in normal group,and the lipid in treated group was less than in model group.The decrement of lipid increased with the increase of drug concentration,which was the highest in CB group,and the effect was most obvious when the CB concentration is 2.5mg/mL.PPARa and PPARγ were detected by real-time PCR,and there was almost no difference between normal and model group.The relative protein expression of PPARa and PPARy were detected by Western Blot method,and there was almost no difference of PPARa between normal and model group.While PPARy of CB group was significantly higher than that of other groups(P<0.05),indicating statistical differences.Conclusion:(1)Theoretical study showed that hyperlipidemia was caused by the the pathogenesis characteristics "phlegm blockage and qi stagnation"(2)This study comfirmed that citrus changshan-huyou peel flavonoids can significantly reduce cholesterol and triglyceride.The expression of PPARa and PPARy under environment of high cholesterol and high oleic acid showed a downward trend,while the expression of them could be restored by the processing of citrus changshan-huyou peel flavonoids,with the level of cholesterol and triglyceride in relative model cells decreased in different degree,which suggested that citrus changshan-huyou peel flavonoids may reduce the level of cholesterol and triglyceride through regulating the expression of PPARa and PPARγ.Totally 4 flavonoids components were extracted and separated from citrus changshan-huyou peel.According to reducing-lipid screening and evaluation model of HepG2 cells in vitro and observation of effectiveness on lipid metabolism and PPARs pathway，CB group was identified as the main active substance playing important role in lipid-lowering function. |
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