| Purpose:This experiment from traditional Chinese medicine "Yin and Yang Freemasons" theory, select of Chinese classic prescription nourishing yin and tonifying kidney fill the essence Zuogui Wan and warming yang and tonifying kidney fill the essence Yougui Wan and its disassembled prescriptions, with the two common medicine for the common medicine group, with Zuogui Wan of drugs for nourishing Yin for the nourishing kidney-yin medicine group, with Yougui Wan of drugs for warming Yang for the tonifying kidney-yang medicine group, to explore protective effect and its mechanism of Zuogui pill, Yougui pill and its demolition part on lipid metabolism disorders in ovariectomized rats, and to explore the relation between the kidney essence deficiency and virtual and lipid metabolism disorders of postmenopausal woman. Material and method:90 SPF 2 month old female reproductive SD rat selected, weight 200~220 g, stratified by body weight were randomly and equally divided into: normal control group(Control), sham operation group(SHAM), ovariectomized group(OVX), the positive control drug progynova group(BJL), Zuogui Wan group(ZGW), Yougui Wan group(YGW), common medicine group(GTY), nourishing kidney-yin medicine group(ZYY), tonifying kidney-yang medicine group(BYY). The ovary groups was injected by intraperitoneal injection with 10% Chlorine hydrate solution 0.3 mL·100 g-1 weight. After the solution of the Chlorine hydrate, in aseptic environment downlink back surgery bilateral ovaries, the sham operated group was treated by the same method, and the right amount of adipose tissue was removed in the sham operation group, and the normal group did not do any treatment. After the operation, rats in each group were given normal drinking water and second days to start free diet. After 1 week, the rats were weighed and began to give medicine. After treatment, the weight of 1 times every 4 weeks. Rats for each kilogram of body weight dose is 6.3 times computation, calculated the daily dose of each drug group: BJL 0.09 mg·kg-1, ZGW 9.45 g·kg-1, YGW 10.26 g·kg-1, GTY 7.56 g·kg-1, ZYY 6.48 g·kg-1, BYY g·kg-1 4.05 g·kg-1. Chinese drug is made into the water decoction, the positive control drug estradiol valerate is made into the suspension liquid by distilled water, all the drug administration groups were given gastric administration, normal control group, sham operation group and ovariectomized group were given distilled water(give 1mL every 100 g weight). After 12 weeks drug treatment rats were removed, due to surgery and intragastric administration of the animal death, 76 animals were included in the results. Fasting but giving water for 24 h before operation, 2 h after the last administration weighing, injected by intraperitoneal injection with 10% Chlorine hydrate solution 0.3mL·100g-1 body weight, with a negative pressure tube for blood from the abdominal aorta, and take the serum with centrifuge after static 2 h. A part was used to measure the serum total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol(HDL-C), alanine amino transferase(ALT), aspartate amino shift enzyme(AST) buy full automatic biochemical analyzer; a part of packing-80 ? for storage, used for the detection of serum estradiol(E2) by enzyme-linked immunosorbent assay(ELISA); take fresh rat liver, and weighed wet weight to prepare for rat the liver index calculation, then take the anterior lobe liver of rats, paraformaldehyde for weeks, paraffin sections were prepared for HE staining for liver morphological detection; take the middle lobe liver of rats placed into a sterile EP tube for-80 ? frozen, for the preparation of frozen sections, the oil red stain was carried out, and the fat accumulation in the liver of ovariectomized rats was observed under the microscope, Spectrophotometric method was established for the determination of liver tissue homogenate lipid peroxide lipid peroxide(LPO), the content of hydrogen peroxide(H2O2), TBA method measured the content of malondialdehyde(MDA), hydroxylamine method, enzyme promoting method, visible light was measured in testing the activity of total superoxide dismutase(T-SOD), glutathione peroxidase(GSH PX), catalase(CAT) of the liver tissue homogenate in ovariectomized rats; take the posterior lobe liver, placed into a sterile EP tube for-80? frozen, a part for detecting gene expression of Lipoprotein lipase(LPL), peroxisome proliferator activation receptor gamma(PPAR-?) in in ovariectomized rats liver tissue by real-time quantitative RT PCR(Q-PCR) and the protein expression of LPL, PPAR-? in in ovariectomized rats liver tissue by Western blot(WB). part of paraformaldehyde soak for a few weeks, made into paraffin sections for detection of positive expression of LPL and PPAR-? in ovariectomized rats liver by immunohistochemistry. Results: 1. Effects of Zuoguiwan,YouguiWan and its demolition part on serum E2 in ovariectomized ratsCompared with the normal group, sham operation group E2 level close to the normal group, ovariectomized group E2 level decreased(P < 0.01), Other groups no significant difference(P>0.05); compared with the ovariectomized group, each treatment group can increase E2 level(P<0.05 or P<0.01); compared with Zuogui Wan group, estradiol valerate group increased E2 level is better than that of Zuogui Wan, but the difference was not statistically significant(P>0.05), Yougui Wan group and common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group are not as good as Zuogui Wan group in terms of increased E2 level(P<0.05). 2. Effects of Zuoguiwan,Yougui Wan and its demolition part on body weight, blood lipid, liver lipid metabolism in ovariectomized rats.There is no difference between the initial body weight of rats in each group, from the beginning of the first week after ovariectomizeding, compared with the normal group, body weight of rats in ovariectomized group increased(P<0.05), indicating the ovariectomized rats weight increased; at fifth weeks, compared with the normal group, ovariectomized group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group rats weight increased(P<0.01 or P<0.05); compared with the ovariectomized group, estradiol valerate group, Zuoguiwan group, rats decreased(P<0.01 or P<0.05); at ninth weeks, compared with the normal group, ovariectomized group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group body weight of rats increased(P<0.01 or P<0.05); compared with the ovariectomized group, estradiol valerate group, Zuoguiwan, YouguiWan group rats weight decreased(P<0.01 or P<0.05); at thirteenth weeks, compared with the normal group, ovariectomized group, estradiol valerate group and common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group rats elevated(P<0.01 or P<0.05); compared with the ovariectomized group, estradiol valerate group, Zuogui Wan group, Yougui Wan group rats body weight decreased(P<0.05 or P<0.05), indicating that Zuogui Wan group, Yougui Wan group on the ovariectomized rats body fat increased inhibitory effect.Compared with the normal group, serum TC, TG and LDL-C were significantly increased in ovariectomized group(P<0.01), the sham operation group close to the normal group, the other groups were increased, but no significant difference(P>0.05), ovariectomized group serum HDL-C decreased significantly(P<0.01), sham operation group close to the normal group, the other groups decreased, but no significant difference(P>0.05); compared with the ovariectomized group, estradiol valerate group, Zuoguiwan group, Yougui Wan group could significantly decrease the serum levels of TC and TG(P<0.01 or P<0.05), each treatment group could significantly decrease the serum LDL-C(P<0.01 or P<0.05), every treatment group significantly increased serum HDL-C(P<0.01), each group had no significant difference(P>0.05). Zuoguiwan, YouguiWan and its disassembled all can improve blood lipid in ovariectomized rats, effects of Zuogui Wan, Yougui Wan are better, Zuogui Wan is better than Yougui Wan, but the difference was not statistically significant.Liver oil red staining showed that in normal group and sham operation group there is a small amount of oil red staining, accumulation of fat is not obvious. the ovariectomized group have more oil red staining, the liver fat accumulation was significantly increased than normal group, estradiol valerate group, Zuogui Wan group, Yougui Wan group oil red staining is less, fat accumulation is not obvious; compared with ovariectomized group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group, the liver oil red staining also decreased, fat accumulation was significantly reduced. Illustrate that ovariectomized rat liver increased accumulation, Zuogui Wan, Yougui Wan and its disassembled can reduce the accumulation of fat in the liver which Zuogui Wan and Yougui Wan effect better. 3 Effects of Zuoguiwan,YouguiWan and its demolition part on liver index in ovariectomized ratsCompared with the normal group, in the sham operation group, the liver index close to the normal group, ovariectomized group, common medicine group, drugs for nourishing Yin group, Yang medicine group, the liver index increased significantly(P<0.01 or P<0.05), estradiol valerate group, Zuogui Wan group, Yougui Wan group were also increased, but the difference was not statistically significant(P>0.05); compared with the ovariectomized group, every treatment group can significantly reduce the liver index(P<0.01); and there is no significant difference among the treatment group(P>0.05). 4 Effects of Zuoguiwan,YouguiWan and its demolition part on liver tissue morphology in ovariectomized ratsThe normal group and sham operation group showed normal structure of liver tissue, liver cells arranged in neat form clear, nuclear membrane integrity, clear nucleoli, central venous sinus, the central vein lumen was normal; the morphology and structure of the central vein and hepatic cells were deformed in ovariectomized group, liver cell arrangement is slightly disordered, the morphology of hepatocytes is not clear. Under the microscope, inflammatory factor, blood sinus becomes small, the central vein lumen; estradiol valerate group, Zuogui Wan group, Yougui Wan group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group liver tissue morphology improved, liver cells arranged in neat form clear, nuclear membrane integrity, clear nucleolus, blood sinus open the central vein, central venous lumen size close to normal. 5 Effects of Zuoguiwan, YouguiWan and its demolition part on antioxidant ability in ovariectomized rat liverCompared with the normal group, ovariectomized group, LPO content increased significantly(P<0.01), sham operation group, estradiol valerate group, Zuo Gui group, Yougui Wan group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group had no significant difference(P>0.05), ovariectomized group, Yougui Wan group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group MDA increased significantly(P<0.01), there was no significant difference in other groups(P>0.05), ovariectomized group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group H2O2 content was significantly increased(P<0.05 or P<0.01), there was no signifycant difference between the other groups(P>0.05); compared with the ovariectomized group, the treatment group LPO, MDA and H2O2 were lower(P<0.05 or P<0.01); compared with Zuoguiwan group, estradiol valerate group decreased the LPO, MDA and H2O2 had no significant difference(P>0.05), Yougui Wan group, the common drug group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group decreased LPO, MDA and H2O2 were less than the effect of Zuogui group(P<0.05 or P<0.01).Compared with the normal group, ovariectomized group T-SOD, GSH-PX activity decreased significantly(P<0.05 or P<0.01), estradiol valerate group, Zuoguiwan group TSOD, GSH-PX activity increased significantly(P<0.01), there was no significant difference between the other groups(P>0.05), ovariectomized group CAT activity decreased signifycantly(P<0.01), Zuoguiwan group CAT activity increased significantly(P<0.01), there was no significant difference between the other groups(P>0.05); compared with the ovariectomized group, the treatment group T-SOD, GSH-PX, CAT activity increased(P<0.05 or P<0.01); compared with Zuoguiwan group, the effect of estradiol valerate group increased the T-SOD, GSH-PX, CAT activity had no significant difference(P>0.05), the effect of Yougui Wan group and common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group increased T-SOD, GSH-PX, CAT activity was significantly are not as good as Zuogui Wan group(P<0.01). 6 Effects of Zuoguiwan, YouguiWan and its demolition part on liver function in ovariectomized ratCompared with normal group, serum ALT and AST increased significantly(P<0.01); compared with ovariectomized group, the treatment group can decrease the level of serum ALT, AST(P<0.01 or P<0.05), the treatment group have no significant difference(P>0.05), indicating that Zuogui Wan, Yougui Wan and its disassembled prescriptions has a certain role in the improvement of ovariectomized rat liver.7 Effects of Zuoguiwan, YouguiWan and its demolition part on gene and protein expression of the lipid metabolism influence factor in ovariectomized rats liverResults of the protein expression by immunohistochemical method showed that LPL, PPAR-? in liver tissue have a region positive expression, and in the cytoplasm of liver cells and nuclei were seen positive expression, but the nucleus of positive expression of weak in the cytoplasm. In normal group and sham operation group, the liver tissue showed higher amounts of cells with positive expression and more yellowish brown coloration, and there are lower amounts cells with positive expression and lower amount of brown coloring in the liver tissue in ovariectomized group, after drug treatment, estradiol valerate group, Zuogui Wan group, Yougui Wan group in liver tissue with a marked increase in the number of positive expression in liver cells, close to the normal group, the common medicine group, nourishing kidney-yin drugs, tonifying kidney-yang drugs the number of cells with positive expression in liver also increased, but not as good as the Zuogui Wan, Yougui Wan group significantly.The results of mRNA expression by Q-PCR method shows that: compared with the normal group, LPL mRNA expression down regulate in ovariectomized group, PPAR-? mRNA expression down regulate in ovariectomized group, PPAR-? mRNA expression upregulate in estradiol valerate group, Zuoguiwan group, Yougui Wan group(P<0.01), the other groups had no significant difference(P>0.05); compared with the ovariectomized group, LPL, PPAR-? mRNA expression upregulated in the sham operation group, estradiol valerate group, Zuoguiwan, YouguiWan group, common medicine group, nourishing kidney-yin drugs, tonifying kidney-yang drugs(P<0.01); compared with Zuogui Wan group, LPL, PPAR-? mRNA expression in estradiol valerate group, Yougui Wan group has no significantly effect(P>0.05), the effect of LPL, PPAR-? mRNA expression of common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group is not asgood as Zuoguiwan group(P<0.01).The results of protein expression by Western Blot method showed that: compared with the normal group, LPL protein expression down regulate in ovariectomized group, PPAR-? protein expression down regulate in ovariectomized group, estradiol valerate group, Zuogui Wan group, Yougui Wan group PPAR-? protein expression increased(P<0.05 or P<0.01), the other groups had no significant difference(P>0.05); compared with the ovariectomized group, LPL, PPAR-? protein expression in sham operation group, estradiol valerate group, Zuogui Wan group, YouguiWan group, common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group increased(P<0.01); compared with Zuogui Wan group, LPL, PPAR-? protein expression in estradiol valerate group, Yougui Wan group has no significantly effect(P>0.05), the effect of LPL, PPAR-? protein expression of common medicine group, nourishing kidney-yin medicine group, tonifying kidney-yang medicine group is not as good as Zuoguiwan group(P<0.01). Conclusion: 1. After removal of bilateral ovaries in rats, estrogen levels is low, weight gain, elevated blood lipids, liver fat accumulation, successfully simulation of postmenopausal lipid metabolism disorder; Zuogui Wan, Yougui Wan and its disassembled prescriptions can increase the estrogen level in ovariectomized rats, and can improve the lipid metabolism in ovariectomized rats; in promoting of estrogen level in ovariectomized rats, the effect of Zuogui Wan is better than that of Yougui Wan, common medicine group, nourishing kidney-yin drugs, tonifying kidney-yang drugs, in improving of lipid metabolism in ovariectomized rats, effects of Zuogui Wan, Yougui Wan are better than that of common medicine group, nourishing kidney-yin drugs, tonifying kidney-yang drugs. So the treatment of postmenopausal women disorder of lipid metabolism can use the method of reinforcing kidney supplying emission, and Ziyin kidney, warming yang and tonifying kidney drugs used in combination is better than pure nourishing yin, pure warming yang and tonifying kidney. 2. After removal of the bilateral ovaries in rats, the liver morphology and structure were deformed, the antioxidant capacity of the liver decreased, and the liver function decreased; Zuogui Wan, Yougui Wan and its disassembled prescriptions can improve liver tissue morphology and enhance liver antioxidant ability, improve the liver function in ovariectomized rat, in terms of enhancing the liver antioxidation ability in ovariectomized rats, Zuogui Wan is better than that of Yougui Wan, common medicine, nourishing kidney-yin medicine, tonifying kidney-yang medicine. 3. After removal of bilateral ovaries in rats, liver LPL, PPAR-? gene and protein expression level was decreased, Zuogui Wan, Yougui Wan and its disassembled party can increase LPL, PPAR-? gene and protein expression levels to improve lipid metabolism in ovariectomized rats liver to improve lipid metabolism in ovariectomized rats, and the effects of Zuogui Wan, Yougui Wan are better than that of common medicine, nourishing kidney-yin medicine, tonifying kidney-yang medicine. 4 Deficiency of kidney essence is one of the causes of the disorder of lipid metabolism in postmenopausal women. |
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