The Role Of SREBP-1c/FAS Signaling Pathway During The Development Of Atherosclerosis Induced By Chronic Intermittent Hypoxia And Insulin Resistance And The Intervention Effect Of Chinese Medicine Of Supplimenting Qi And Activating Blood Circulation

Obstructive sleep apnea-hypopnea syndrome(OSAHS),which has high morbidity,is an independent risk factor for cardiovascular diseases and may result in hypertension,myocardial infarction,heart failure and even sudden death during the night.Chronic intermittent hypoxia(CIH)caused by OSAHS,could lead to the deterioration of the insulin resistance(IR)and the formation of glucose and lipid metabolic disorder.The synergy of CIH and IR can promote vascular endothelial injury and cause OSAHS related atherosclerosis(AS)and other vascular lesions.Some studies have found that SREBP-lc/FAS signaling pathway plays an important role in the process of CIH composite IR mediated vascular endothelial injury and AS.In the theory of traditional Chinese medicine(TCM),AS often falls to the category of "Qi deficiency and blood stasis" syndrome.Previous studies suggested that Chinese herbal medicine for supplimenting Qi and activating blood circulation have the effects of anti-imflammatory,IR-improving,and vascular endothelial protecting.The therapeutic principle was found to be clinical effective in OSAHS related AS.However,the potential mechanism of these functions is not yet revealed.This study took CIH composite IR mediated AS as a target,and used ginsenoside and ligustrazine under the guidance of TCM theory.This study built a model system of CIH composite IR,both in vitro and in vivo,to observe the molecular mechanism about SREBP-lc/FAS signaling pathway and the intervention effect of Chinese herbal medicine for supplimenting Qi and activating blood circulation.This paper was composed of two main parts:1.Literature reviews:Including the following two parts:Prevention and treatment of obstructive sleep apnea hypopnea syndrome related cardiovascular diseases in theory of integrated Chinese and western medicine;Roles and mechanisms of chronic intermittent hypoxia in atherosclerosis.Then systematically reviewed the efficacy and safety of ginseng preparations in improving IR in the third part.The Meta-analysis suggested that compared with non-TCM treatment,standard treatment of Western medicine or conventional treatment,treatment combined with ginseng herb,ginseng extract and its compound Chinese medicinal formulae could improve insulin sensitivity and reduce blood glucose in IR patients with type 2 diabetes and metabolic syndrome.Rare adverse reactions occurred and safety evaluation was acceptable.2.Experimental research:Including the following four parts:Study 1:Establishment of a cell model of intermittent hypoxia and insulin resistance and the effect of intermittent hypoxia on SREBP-1c/FAS signaling pathway.Objective:To establish intermittent hypoxia(IH)composite IR cell model of 3T3-L1 adipocytes in vitro,and to observe the effect of IH combined with IR on the SREBP-1c/FAS signaling pathway.Methods:3T3-L1 preadipocytes were differentiated into mature 3T3-L1 adipocytes;the latter were treated with complete culture medium containing DEX 1×106mol/L for 96h to establish IR-3T3-L1 adipocyte model.Then IR-3T3-L1 adipocytes were treated with IH 0 cycles,2 cycles,4 cycles,8 cycles,16 cycles,32 cycles,and SH group was treated with sustained hypoxia.Methods of qPCR was used to detect mRNA expression level of SREBP-lc,FAS and IRS-1.Western-blotting were used to detect protein expression level of SREBP-1 and FAS.Results:1.After differentiated for 8-11 days,the differentiation rate of 3T3-L1 cells exceeded 80%,and 3T3-L1 preadipocytes were successfully differentiated into mature 3T3-L1 adipocytes.2.DEX treatment of differentiated mature 3T3-L1 adipocytes after 96h,medium glucose was higher than the control group,IRS-1 mRNA expression level was lower than the control group,P<0.05.3.Within 32 cycles of IH,IR-3T3-L1 adipocytes’ mRNA and protein expression level of SREBP-lc,FAS were increased with the increasing of IH cycles;the mRNA expression of IRS-1 was decreased after IH 32 cycles,P<0.05,and lower than that of SH group,P<0.05.Conclusion:Within a certain range,IH could promote the expression of SREBP-lc/FAS signaling pathway of IR-3T3-L1 adipocytes,and the expression level has a positive correlation to the number of IH cycles.And IH has the effect of promoting IR in vitro.Study 2:The intervention effects and the mechanism of monomers of Chinese medicine supplimenting Qi and activating blood circulation on IH composite IR cell model.Objective:To observe the intervention effects of ginsenoside Re combined with Ligustrazine(TMP)on IH composite IR cell model of 3T3-L1 adipocytes in vitro,and to observe the effect of SREBP-lc/FAS signal pathway.Methods:IH composite IR cell model of 3T3-L1 adipocytes were built.For screening of suitable intervention concentration of ginsenoside Re and TMP,IR-3T3-L1 adipocytes were treated in cell culture medium containing ginsenoside Re 0μmol/L,1μmol/L,10μmol/L,100μmol/L,500μmol/L and TMP 0μmol/L and 1μmol/L,10μmol/L,100μmol/L,1000μmol/L,10000μmol/L.Cell viability were detected by CCK-8 assay,and the expression level of SREBP-1c and FAS mRNA were detected by qPCR.After selecting of effective concentration of inhibiting SREBP-lc and FAS,the following four kinds of Re+TMP combinations were used:Re 1μmol/L + TMP 1μmol/L,Re 1μmol/L+TMP10μmol/L,Re 10μmol/L + TMP 1μmol/L,Re 10μmol/L + TMP 10μmol/L.qPCR was used to detect the expression level of mRNA of SREBP-1c and FAS to screen the optimal concentration of Re+TMP.Then IR-3T3-L1 adipocytes were divided into normoxia control group(Con),model group(IH),SREBPs inhibitor group(Betulin)and traditional Chinese medicine group(Re+TMP).With pretreatment for 24h of corresponding drugs respectively,IH group,Betulin group and Re+TMP group were treated by IH for 32 cycles.Western-blotting was used to detect the protein expression of SREBP-1 and FAS,and qPCR was used to detect the expression level of IL-6,SOD,IRS-1 and HIF-1α.Results:1.There was no statistically significant difference between the four different concentrations of ginsenoside Re effects on cell viability,P>0.05;Re concentration of 1μmol/L,10μmol/L inhibited IH induced upregulation of SREBP-lc mRNA expression,P<0.05;for effects of different concentrations of TMP on cell activity,the concentration of 10000μmol/L reduced cell activity,P<0.05;TMP concentration of 1μmol/L,10μmol/L inhibited IH induced upregulation of SREBP-1c mRNA expression,P<0.05.2.The concentration of Re 1μmol/L + TMP 10μmol/L showed the most obvious inhibition of the expression of SREBP-1c and FAS mRNA after IH treatment,P<0.05.3.Betulin and Re+TMP could inhibite IH induced upregulation of protein expression of SREBP-1 and FAS,P<0.05,increase the mRNA expression level of IRS-1 and SOD after IH treatment,P<0.05,and reduce the mRNA expression level of HIF-la after IH treatment,P<0.05.Conclusion:The combination of ginsenoside Re and TMP has the effect of inhibiting expression of IH promoted SREBP-lc/FAS pathway in IR-3T3-L1 adipocytes,and may have the effect of improving IR and oxidative stress.Study 3:Establishment of an animal model of chronic intermittent hypoxia composite insulin resistance ApoE-/-mice model and the effect of chronic intermittent hypoxia on SREBP-1c/FAS signaling pathway.Objective:To establish an ApoE-/-mice model of CIH combined with IR inducing AS,and observe the effect of SREBP-lc/FAS signal pathway.Methods:ApoE-/-mice were fed with high-fat diet for four weeks then intraperitoneal injected of STZ solution to establish IR-ApoE-/-mice model.A totle of 16 IR-ApoE-/-mice were randomly and averagely divided into normoxic control group(NA)and model group(CIH)which were fed with high-fat diet.IR-ApoE-/-mice of CIH group were treated with IH,which meaned the oxygen concentration falled to 10±0.5%in the first 90 seconds of one cycle and then increased to 21±0.5%in the later 90 seconds,continuous for eight hours per day(09:00-17:00)and eight weeks.Eight C57BL/6J mice were used as blank control group(Con)which was fed with conventional diet.The mice were weighed and tested of fasting blood glucose every four weeks.At the end of the experiment,serum insulin level was measured by ELISA assay.Oil red O staining was used to compare the plaque of the aorta in each group.Methods of qPCR and Western-blotting were used to detecte SREBP-1c,FAS mRNA and protein expression levels of the aorta,skeletal muscle and liver,as well as IRS-1 mRNA expression and protein level of p-IRS-1.Results:1.After CIH treatment of 4w and 8w,fasting blood glucose of NA group,CIH group was higher than Con group,P<0.05;and insulin level of CIH group was higher than Con group after CIH treatment of 8w,P<0.05.2.The expression of IRS-1 mRNA level in aorta,skeletal muscle and liver of CIH group were lower than those in Con group,P<0.05.3.Oil red O staining showed obvious AS aortic plaque in CIH group.4.Comparison of the mRNA expression level of SREBP-lc and FAS,expression of SREBP-lc,FAS mRNA in aorta,skeletal muscle and liver were significantly higher in CIH group than those in Con group,P<0.05;while comparison of the protein level of SREBP-1 and FAS,expression of SREBP-lc,FAS protein in aorta,skeletal muscle were significantly higher in CIH group than those in Con group,P<0.05.Conclusion:In ApoE-/-mice,synergy of CIH and IR could upregulate SREBP-1c/FAS signaling pathway expression in aorta,skeletal muscle and liver,and might promote IR and ultimately promoted AS.Study 4:The intervention effects and the mechanism of Chinese herbal medicine of supplimenting Qi and activating blood circulation on chronic intermittent hypoxia composite insulin resistance ApoE-/-mice model.Objective:To observe the intervention effects of ginsenosides combined with TMP on CIH composite IR ApoE-/-mice model in vivo,and to observe the effect of SREBP-1c/FAS signaling pathway.Methods:CIH composite IR ApoE-/-mice model were built.70 IR-ApoE-/-mice were randomly divided into normoxic control group(NA),model group(CIH)and SREBPs inhibitor group(Betulin),atorvastatin group(WM),TCM low-dose group(TCM-L),TCM middle-dose group(TCM-M)and TCM high-dose group(TCM-H)group with 10 rats in each group.Before IH treatment everyday,these mice were intragastric administrated respectively with normal saline,Betulin,atorvastatin,ginsenosides + TMP of low,medium and high dose for eight weeks.Besides,10 C57BL/6J mice were used as blank control group(Con).The mice were weighed and tested of fasting blood glucose every four weeks.At the end of the experiment,serum insulin level and C-reactive protein(CRP)was measured by ELISA assay,the serum total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and superoxide dismutase(SOD)levels were measured in method of colorimetric.Oil red O staining was used to compare the plaque of the aorta in each group.Methods of qPCR and Western-blotting were used to detect SREBP-1c,FAS mRNA and protein expression levels of the aorta,skeletal muscle and liver.Inflammatory cytokines of aorta including TNF-a,IL-6,CD106(VCAM-1)mRNA and protein expression levels were observed.Aorta,skeletal muscle and liver IRS-1 mRNA expression and protein level of p-IRS-1,expression levels of HIF-la of aortic and skeletal musclem were observed as well.Results:1.During experimental process,two mice were died in CIH group,one in Betulin group,one in TCM-L group and one in TCM-M group.At the end of the experiment,mice of WM group and TCM-H group were lighter than the NA group,CIH group and Con group,P<0.05;fasting blood glucose in TCM-H group was decreased than other experimental groups,P<0.05;the difference of insulin levels amang groups was not statistically significant,P>0.05.For serum lipid levels,TC,TG and LDL-C in CIH group were higher than that of Con group,P<0.05;HDL-C level in CIH group was lower than Con group,P<0.05;HDL-C level in Betulin group,WM group,TCM-H group was higher than CIH group,P<0.05;LDL-C level in TCM-M group,TCM-H group is lower than the CIH group,P<0.05.The level of CRP in CIH group was higher than other groups,P<0.05.The level of SOD was lower in CIH group than Con group,NA group,P<0.05,TCM-H group was higher than CIH group,P<0.05.2.Oil red O staining showed proximal aorta of AS plaque results.NA group and CIH group showed obvious AS aortic plaque,while Betulin group,WM group,TCM-H group showed AS plaque reduction.For thoracic aorta and abdominal aorta,AS plaque in CIH group were multiple and the area is large,while that in WM group and TCM-H were less and smaller than CIH group.3.Comparison of the expression level of SREBP-1c/FAS signaling pathway in tissues,expression of SREBP-1c,FAS mRNA and protein levels in aorta,skeletal muscle and liver were significantly higher in CIH group than those in Con group,P<0.05;SREBP-1c,FAS mRNA and protein expression in aorta and skeletal muscle in TCM-H group were lower than CIH group,P<0.05.4.Aortic inflammation cytokines including IL-6,TNF-α,and CD 106(VCAM-1)expression level were higher in CIH group than Con group,P<0.05;in TCM-H group,TNF-a,IL-6,CD 106(VCAM-1)mRNA expression levels were lower than that in CIH group,P<0.05,TNF-a,CD 106(VCAM-1)protein expression levels were reduced compared with CIH group,P<0.05.5.The expression of IRS-1 mRNA levels in aorta,skeletal muscle and liver tissues of CIH group were lower than that in Con group,P<0.05;compare the level of p-IRS-1 protein,aorta,skeletal muscle and liver tissue of TCM-H group were significantly higher than those in CIH group,P<0.05.6.Expression of HIF-1α level in aorta and liver of CIH group were elevated compared to Con group,P<0.05;in aorta,liver of TCM-H group,the expression of mRNA and protein of HIF-1α were lower than CIH group,P<0.05.Conclusion:Ginsenosides and TMP combination,which was chosen as representative of Chinese medicine of supplimenting Qi and activating blood circulation,could improve IR and serum lipid levels,inhibiting inflammation and oxidative stress,and ultimately alleviate AS to some extent.And the mechanism of its intervention effects might be related to the inhibition of CIH promoted upregulation of SREBP-lc/FAS signaling pathway.