| The disorders of lipid metabolism in postmenopausal women were closely related to the decrease of estrogen level,and Estrogen Replacement Therapy(ERT)was commonly used in clinic.Pharmacological studies have shown that estrogen can improve dyslipidemia,but large-scale clinical trials have shown that ERT would increase the incidence of stroke,venous thrombosis and breast cancer,therefore,ERT was not desirable.Traditional Chinese medicine believed that menopause was a manifestation of the decline of kidney qi and kidney essence deficiency,so it is very important to regulate the Yin and Yang of perimenopausal syndrome.As traditional Chinese medicine,Semen Astragali Complana(SAC)has the function of tonifying kidney and strengthening essence,which can be concluded that warm kidney yang can help to achieve the effect of reducing blood lipid,and modern research showed that it has significant lipid-lowering effect,especially to reduce the effect of TG.SAC have a better lipid regulating effect,and the mechanism was related to estrogen receptor,but was significant difference from ERT.Therefore,in this study,the metabolic pathway of TG was used as the breakthrough point,we can investigate the effect of lipid lowering and estrogen receptor in liver from the extract of SAC by hyperlipidemia rats with kidney-yang deficiency pattern induced by ovariectomy and high-fat feeding,analyse the differences between the Chinese medicine and ERT,clarify the characteristics and advantages of regulating blood lipid by Yang medicine Tonics,investigate the mechanism of lipid of SAC with the function of warming kidney yang to resolve turbidity,enrich the scientific connotation of the theory of Yang Tonics.ObjectiveTo investigate the effect of lipid metabolism mediated by liver estrogen receptor on extract from SAC and Total Flavonoids of Astragalus complanatys(TFAC)via TG synthesis pathway,transport pathway and hydrolysis pathway in hyperlipidemia model of kidney yang deficiency model rat.Methods1.Preparation and content determination of extract from SAC.2.To investigate the lipid metabolism mediated by liver estrogen receptor from the extract of SAC in model rats by testing the level of body weight,TG,TC,LDL-C,HDL-C,E2 and FSH in serum and expression of ERα mRNA,ERβ mRNA in liver.3.Preparation and content determination of TFAC.4.To investigate the lipid metabolism mediated by liver estrogen receptor from TFAC in model rats by testing the level of body weight,liver weight,uterus weight and TG,TC,LDL-C,HDL-C,E2 and FSH in serum and the expression of ERa protein in liver.5.To investigate the effects of TFAC on TG synthesis pathway in model rats by testing the level of ACC,FAS,GPAT,CPT-1α,ACO in liver and the expression of SREBP-1c,PPAR protein in liver.6.To investigate the effects of TFAC on TG transport relational pathway in model rats by testing the level of ApoB 100,VLDL in serum and the expression of HNF-4a、MTP protein in liver.7.To investigate the effects of TFAC on TG hydrolysis relational pathway in model rats by testing the expression of ATGL,HSL,PLIN,PPAR γ protein in liver.ResultsPart 1Effects of extract from SAC in lipid regulating effects mediated by estrogen receptor.(preventive administration)1.1 Preparation and content determination of extract from SACExtract of SAC was the substance after the decoction and dry by oven of SAC,measured the extract rate was about 19.8%.1.2 Effect of extracts from SAC on body weight in model ratsThe body weight of model group after giving high fat diet began to increase significantly from the first week(P<0.05).The body weight of estrogen group and high dose group of extracts from SAC(SACH)after giving high fat diet began to decrease significantly from the fifth week(P<0.05)1.3 Effect of extracts from SAC on serum level of TG in model ratsThe serum level of TG of model group after giving high fat diet began to increase significantly from the sixth week(P<0.05).The serum level of TG of estrogen group and SACH group after giving high fat diet began to decrease significantly from the sixth week(P<0.05)1.4 Effect of extracts from SAC on serum level of TC in model ratsThe serum level of TC of model group after giving high fat diet began to increase significantly from the fifth week(P<0.01).The serum level of TC of estrogen group and the middle dose group of extracts from SAC(SACM)after giving high fat diet began to decrease significantly from the sixth week(P<0.05).The serum level of TC of SACH group began to decrease significantly from the seventh week(P<0.05).1.5 Effect of extracts from SAC on serum level of HDL-C in model ratsThe serum level of HDL-C of model group after giving high fat diet began to decrease significantly from the seventh week(P<0.05).The serum level of HDL-C of estrogen group and SACM group after giving high fat diet began to increase significantly from the sixth week(P<0.05).The serum level of HDL-C of the low dose group of extracts from SAC(SACL)and SACH group after giving high fat diet began to increase significantly from the eighth week(P<0.05).1.6 Effect of extracts from SAC on serum level of LDL-C in model ratsThe serum level of LDL-C of model group after giving high fat diet began to increase significantly from the fifth week(P<0.01).The serum level of LDL-C of estrogen group after giving high fat diet began to decrease significantly from the sixth week(P<0.05).The serum level of LDL-C of SACM group after giving high fat diet began to decrease significantly from the seventh week(P<0.05).The serum level of LDL-C of SACH after giving high fat diet began to decrease significantly from the eighth week(P<0.001).1.7 Effect of extracts from SAC on serum level of E2 and FSH in model ratsThe serum level of E2 decreased significantly(P<0.01),the serum level of FSH of model group increased significantly(P<0.05).The serum level of E2 increased significantly(P<0.05),the serum level of FSH of estrogen group decreased significantly(P<0.05).There was no statistical difference in serum level of E2 and FSH among the rats in SACL,SACM and SACH group(P>0.05).1.8 Effect of extracts from SAC on the expression of ERα mRNA in liver in model ratsThe expression of ERa mRNA in liver of model group decreased significantly(P<0.05).The expression of ERa mRNA in liver of estrogen group,SACL group,SACM group,SACH group increased significantly(P<0.05).ConclusionThe experimental result showed that hyperlipidemia rat with kidney-yang deficiency pattern was successfully established after 5-7 weeks high fat diet.By the way of prophylactic administration,the extract of SAC can regulate the disorder of blood lipid of model rats by warming kidney yang to resolve turbidity,the mechanism may not raise the serum level of E2 in model rats,but may increase the sensitivity of the estrogen receptor or increase the number of estrogen receptors and post receptor signal amplification,produce the effect of estrogen and regulating blood lipid status.On the basis,by analyzing the effective components of SAC,we can further study the material basis and the mechanism of lipid lowering effect.The composition and content of extracts from SAC was uncertain,so that further research was lack the basis.The study showed that TFAC were the main chemical composition,therefore,we took TFAC as the research focus,for further study the mechanism of reducing blood lipids.Part 2Effects of FAC on lipid regulating mechanism mediated by estrogen receptor.(therapeutic administration)2.1 Extraction and content determination of FACThe FAC was extracted by macroporous resin and decoction,measured the extract rate was 3.8%.2.2 Lipid regulating effects of FAC in hyperlipidemia rat with kidney-yang deficiency pattern mediated by estrogen receptor2.2.1 Establishment and evaluation of hyperlipidemia rat with kidney-yang deficiency patternHyperlipidemia rats with kidney-yang deficiency pattern induced by ovariectomy and high-fat feeding,the results showed that the body weight of model group after giving high fat diet began to increase significantly from the first week(P<0.05).The serum level of TG,TC,and LDL-C of model group after giving high fat diet began to increase significantly from the sixth week(P<0.05).2.2.2Effect of FAC on body weight in model ratsThe body weight in model group was significantly increased before administration(P<0.001).After administration,the body weight in model group was significantly increased(P<0.001),the body weight in estrogen group and SACM group were significantly decreased(P<0.05).2.2.3Effect of FAC on liver weight and index in model ratsThe liver weight and index in model group were significantly increased(P<0.05).The liver weight were decreased in estrogen group(P<0.05),the liver weight and index were decreased in SMCH group(P<0.05).2.2.4Effect of FAC on serum level of TG,TC,LDL-C and HDL-C in model ratsThe serum level of TG,TC,LDL-C were significantly increased(P<0.001),the serum level of HDL-C in model group was significantly decreased(P<0.001).The serum level of TG,TC,LDL-C were significantly decreased(P<0.001),the serum level of HDL-C in estrogen,XueZhiKang(XZK)and SMCH group were significantly increased(P<0.05).2.2.5Effect of FAC on liver lipid in model ratsThe level of TG,TC and LDL-C in liver were increased(P<0.001),the level of HDL-C from model group was decreased(P<0.001).The level of TG,TC and LDL-C in liver were decreased(P<0.05),the level of HDL-C was increased from estrogen and XZK group(P<0.05).The level of TC in liver was decreased from SMCL group(P<0.001).The level of TC,LDL-C in liver was decreased from SMCM group(P<0.05).The level of TG,TC and LDL-C in liver were decreased(P<0.05),the level of HDL-C was increased from SMCH group(P<0.05).2.2.6 Effect of FAC on uterine weight and index in model ratsUterus weight and index in model group were significantly decreased(P<0.001).Uterine weight and index were significantly increased from estrogen group(P<0.001).There was no statistical difference in uterus weight and index among the rats in XZK,SMCL,SMCM and SMCH group(P>0.05).2.2.7 Effect of FAC on Serum level of E2,FSH in model rats.The serum level of E2 was significantly decreased(P<0.05),the serum level of FSH from model group was increased(P<0.001).The serum level of E2 from estrogen group was increased(P<0.05),the serum level of FSH was decreased from estrogen group and SACM(P<0.05).There was no statistical difference in serum level of E2 and FSH among the rats in SACL,SACM and SACH group(P>0.05).2.2.8 Effect of FAC on expression of ERa protein in liver in model rats.Immunohistochemistry was used to detect the expression of ERα protein in liver.Results showed that the expression of ERα protein in membrane of liver cell was decreased from model group(P<0.001),the expression of ERa was significantly increased in estrogen group and SACM group(P<0.05).2.3 Effect of FAC on related pathways about TG synthesis in model rats.2.3.1 Effect of FAC on TG synthesis pathway in model rats.The level of ACC,FAS,GPAT in liver were increased significantly in model group(P<0.001).The level of ACC,FAS,GPAT in liver decreased significantly from estrogen group(P<0.05).The level of FAS,GPAT in liver decreased significantly from XZK group(P<0.05).The level of ACC,GPAT in liver decreased significantly from SMCM group and SMCH group.Immunohistochemistry was used to detect the expression of SREBP-1c protein in liver.Results showed that the expression of SREBP-1c protein in membrane of liver cell was increased from model group(P<0.05),the expression of SREBP-1c was significantly decreased in XZK group and SACH group(P<0.05).2.3.2 Effect of FAC on Fatty acid β oxidation pathway in model rats.The level of CPT-1α,ACO in liver were decreased in model group(P<0.05).The level of ACO in liver was increased in estrogen group and XZK group(P<0.05).The level of ACO and CPT-1α in liver were decreased in SACM group and SACH group(P<0.05).Immunohistochemistry was used to detect the expression of PPARa protein in liver.Results showed that the expression of PPARa protein in nuclei of liver cell was decreased from model group(P<0.001),the expression of PPARa protein was significantly increased in estrogen group,SACL group,SACM group and SMCH group(P<0.01).2.4Effect of FAC on TG transport pathway in model ratsThe serum level of ApoB100 and VLDL were decreased in model group(P<0.05).The serum level of ApoB100 was increased significantly in SMCL group and SMCH group(P<0.05).The serum level of VLD was increased significantly in estrogen group and SACM,SACM,SMCH group(P<0.05).Immunohistochemistry was used to detect the expression of MTP protein in liver.Results showed that the expression of MTP protein in membrane of liver cell was increased from model group,the expression of MTP protein was increased in estrogen,SACL,SACM and SMCH group(P<0.05).2.5Effect of FAC on TG hydrolysis pathway in model ratsImmunohistochemistry was used to detect the expression of PPARγ、ATGL、HSL、PLIN protein in liver.Results showed that the expression of PPARγ、ATGL、HSL、PLIN protein in membrane of liver cell was decreased from model group(P<0.001),the expression of PPARγ、ATGL、HSL、PLIN protein was increased in estrogen,SACL,SACM and SMCH group(P<0.05).ConclusionThe experimental result showed the stability and reproducibility of the hyperlipidemia rat with kidney-yang deficiency pattern.Further study showed that the TFAC was the main effective component of lipid-lowering effect of SAC.TFAC may not improve the serum level of E2,but may enhance the sensitivity of the estrogen receptor or increase the number of estrogen receptors and post receptor signal amplification,which may regulate different target and achieve the lipid-lowering effect.TFAC has significant effects in reducing the level of TG,which can act on the pathway for the synthesis,transport and hydrolysis of TG.TFAC can inhibit the expression of SREBP-lc in liver,reduce the level of ACC in fatty acid synthesis process and GPAT in TG synthesis process,and decrease the rate of TG,at the same time,TFAC can raise the expression of PPARa in liver,increase the level of CPT-1α and ACO in fatty acid beta oxidation pathway,accelerate the rate of pathway,and reduce the deposition of TG in liver,both can inhibit the synthesis of TG in liver.TFAC can raise the expression of MTP in liver,accelerate the assembly and secretion of ApoB100 and VLDL in serum,promote the output of VLDL-TG,and increase hepatic TG entering into bloodstream.TFAC can raise the expression of PPARy in liver,increase the expression of ATGL,HSLand PLIN protein in liver,reduce the deposition of TG in liver.All those can reduce the level of TG Estrogen can reduce the level of ACC and FAS in fatty acid synthesis process and GPAT in TG synthesis process and decrease the rate of TG in liver,at the same time,estrogen can raise the expression of PPARα in liver,increase the level of ACO in fatty acid beta oxidation pathway,accelerate the rate of pathway,and reduce the deposition of TG in liver,both can inhibit the synthesis of TG in liver.Estrogen can accelerate the transport pathway of TG,promote the output of VLDL-TG,increase TG in liver transport into blood.The three pathways can reduce the serum of TGExperimental research showed that the lipid-lowering effect of SAC and estrogen,which can reduce the serum level of TG,TC and LDL-C,increase the serum level of HDL-C,but the effect of SAC on serum HDL-C is stronger than estrogen and the targets were different.The level of HDL-C was closely related to kidney yang.Therefore,SAC can warm kidney yang,promote qi hua,replete kidney qi,prevent the formation of plaque in the arteries,reduce the risk of cardiovascular disease,but also can avoid the side effect brought by the use of estrogen,further reveals the characteristic and advantage of Chinese herbal medicine "Warm kidney yang to resolve turbidity" in regulating blood lipid,improve the theoretical basis of modern science,which has a great value of clinical application. |
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