| Objective:To evaluate the lipid regulating effects of ShungHua granules on normal mice and different animal models of hyperlipidemia, as well as the lipid peroxidation, lipid metabolism, hemorheology, hepatic histopathology changes and LDLR, PPARy protein expression in hepatic cells of rats, to explore the mechanism of molecular biology. It will be reported a new TCM health products and provided the research article of pharmacokinetics before clinic.Methods:1. The effect of Shuanghua granules on blood lipid in normal mice40 mice were randomly divided into 5 groups as follow:solvent group, Shuanghua granules 22.5 g·kg-1,11.3 g·kg-1 and 5.6 g·kg-1 (according to the recommend for clinic of 60,30,15 times), and were taken intragastric administration once a day, at the same time, solvent group was administered distilled water. The mice serum TC, TG and HDL-C level were determined.2. The effect of Shuanghua granules on blood lipid in acute hyperlipidemia induced with yolk in mice60 mice were randomly divided into normal group, model group, Zhibituo 2 g·kg-1 group and Shuanghua granules 22.5 g·kg-1,11.3 g·kg-1,5.6 g·kg-1 group,10 mice for each group. After continuous intragastric administration for 3 weeks (normal and model group were administered with distilled water), they were weighed once a week. After 5 h of the 20 d administrations, except the normal group, other groups were ip.75% yolk 20 mL·kg-1, after fasted overnight for 10 h, mice were administered one time again, the serum TC, TG and HDL-C values were determined 2 h later.3. The effect of Shuanghua granules on blood lipid in endogenous hyperlipidemia induced with WR-1339 in mice60 mice were randomly divided into 6 groups, the administration methods same to 2. Positive group was intragastric administration atorvastatin 0.0033 g·kg-1. After continuous intragastric administration for 3 weeks (normal and model group were fed with distilled water), after 1 h at the last administration, except the normal group, other groups were ip. Triton WR-1339 400 mg·kg-1 to induce endogenous hyperlipidemia. 18 h later, serum TC, TG, HDL-C and LDL-C values were determined.4. The effect of ShuangHua granules on blood lipid metabolism and its related genes expression in hyperlipidemia rats60 male rats were randomly divided into 6 groups:normal group, model group and the lovastatin 0.02 g·kg-1 group, Shuanghua granules 7.5 g·kg-1,3.8 g·kg-1,1.9 g·kg-1 group (according to the recommend for clinic of 20,10, and 5 times). Rats were administered at the same time, except the normal group, the other 5 groups were fed high fat diet (78.8% basal diet,10% lard,10% yolk powder,1% cholesterol,3# bile salts 0.2%).3 d before the end of the experiment the rats were be placed in separate metabolic cages, fecal stool were collected for determining total lipid content. After the last administration, rats were fasted 12 h, serum TC, TG, HDL-C, LDL-C, MDA, SOD, NEFA, LPL, HL, LA and hepatic TC, TG, HDL-C, LDL-C levels were determined.4 mL anticoagulant was used to detect hemorheology; a portion of left lobe liver was used to HE detection. The hepatic LDLR, PPARy protein expressions were observed by Western Bloting method.Results:1. The effect of ShuangHua granules on serum TC, TG, HDL-C in normal miceCompared with the control group, ShuangHua granules 22.5,11.3,5.6 g·kg-1 could reduce the serum TG levels (P<0.05, P<0.0), with dose-dependent trend which has more effects in lower dose, TC, HDL-C levels were not significant changed (P> 0.05). ShuangHua granules 45 g·kg-1 had no significant effect on serum TC, TG, HDL-C levels (P> 0.05).2. The effect of Shuanghua granules on serum TC, TG, HDL-C in acute hyperlipidemia induced with yolk in miceThe results showed that compared with normal group, the model group could remarkably increase the serum TC, TG lever (P< 0.01), it means an acute hyperlipidemia model was reproduced successfully. Compared with model group, ShuangHua granules 11.3,5.6 g·kg-1 could significantly reduce TC lever (P< 0.01), there was no significant effects on TG and HDL-C; ShuangHua granules 22.5 g·kg-1 could decrease TC, TG levels, raise HDL-C level, but this change had no significant difference (P> 0.05).3. The effect of Shuanghua granules on serum TC, TG, HDL-C, LDL-C in endogenous hyperlipidemia induced with WR-1339 in miceModel group serum TG, TG, LDL-C were significantly higher than normal group (P< 0.01), it suggested that endogenous hyperlipidemia model was reproduced successfully. Compared with model group, ShuangHua granules 22.5 g·kg-1 could significantly reduced LDL-C levels (P< 0.05). There had no significant effect on TC, TG and HDL-C levels (P> 0.05).4. The effect of Shuanghua granules on serum TC, TG, HDL-C, LDL-C in prey hyperlipidemia rats.Compared with the normal group, SD rats were fed on high fat diet, serum TC, LDL-C abnormally increased and HDL-C decreased (P< 0.01), it showed that prey hyperlipidemic in rats reproduced successfully. Compared with model group, ShuangHua granules can significantly reduce hyperlipidemia rats serum TC, LDL-C levels, increase HDL-C values(P< 0.05, P< 0.01). It indicated that ShuangHua granules can regulate blood lipid levels, then prevent the occurrence and development of hyperlipidemia.5. The effect of Shuanghua granules on hepatic TC, TG, HDL-C, LDL-C in prey hyperlipidemia rats.Model group TC, TG levels significantly increased compared with normal rats (P< 0.05). Compared with model group, ShuangHua granules can significantly reduce the hyperlipidemic rats hepatic TC, TG, LDL-C levels, increase HDL-C values (P< 0.05, P < 0.01).It shows that ShuangHua granules could regulate lipid levels in hyperlipidemic rats liver.6. The effect of Shuanghua granules on serum SOD, MDA, NEFA in prey hyperlipidemia rats.Compared with normal group, model group serum MDA and NEFA were significantly increased and SOD activity was significantly decreased, it indicated that hyperlipidemia rat associated with free radical lipid peroxidation. Compared with model group, ShuangHua granules groups could significantly increase serum SOD level and decrease NEFA level, and 7.5 g·kg-1,3.8 g·kg-1 groups could reduced MDA level (P < 0.05, P< 0.01). It showed that ShuangHua granules have the role of lipid peroxidation and inhibitting the serum NEFA of hyperlipidemia.7. The effect of Shuanghua granules on serum LPL, HL, LA activity and lipid content of feces in prey hyperlipidemia ratsThe results showed that rats fed on high fat diet lead to serum LPL, LA activity decreased, ShuangHua granules could effectively increase LPL, LA and HL levels(P< 0.05, P< 0.01). The lipid content in feces of model rats increased significantly than normal rats (P< 0.05). ShuangHua granules groups' lipid content of feces was no significant difference compared with the model group (P> 0.05).8. The effect of Shuanghua granules on whole blood viscosity, RV andηP in prey hyperlipidemia ratsThe results showed that model group whole blood viscosity andηP were significantly increased than the normal group (P< 0.05, P< 0.01), it showed that hyperlipidemic rats had hemorheology changes. Compared with model group, ShuangHua granules and lovastatin could effectively reduce blood viscosity, and ShuangHua granules 7.5 g·kg-1,3.8 g·kg-1 could also significantly decreasedηP degree (P< 0.05, P< 0.01).9. The effect of Shuanghua granules on HCT, IEA and IED in prey hyperlipidemia rats Compared with the normal group, model group rats IEA, IER significantly increased (P<0.01), and IED was significantly decreased (P<0.01). Compared with model group, ShuangHua granules groups and the lovastatin group can significantly reduce the IEA and the IER levels (P<0.01), but it had no obvious improvement on the HCT and IED (P>0.05).10. The effect of Shuanghua granules on pathomorphology of hepatic tissue in prey hyperlipidemia ratsVisible:The shape of liver was integrated, freshred,capsule smooth and glossy in the normal group; the liver of the model group was soft, slicing greasy which volume and weight of increases, envelope strain, edge bluntness, there was significant difference compared with the normal group; lovastatin and the ShuangHua granules 1.9 g·kg-1 group rats liver were improved compared with model rats.the other groups had no significant difference in involume, weight, quality, color with the model group。The observation of microscope:The liver tissue in the normal group is complete and clear hepatic lobufe's construction is normal,the liver cell arranges orderly around the central vein, The same size and circle liver cell uncleus was clear, located the centre of polygonal cell, cytoplasm is abundant and afterbirth liquid is well-distributed。The hepatic construction disappears in the model group rat, hepatic lobule's boundary is not clear and arranges disorderly, mostly liver sinusoids disappears, There was many and different size lipid droplet vacuoles(fatty degeneration)in the expanded livers cell, as well as inflammatory cell intiltration, HE staining cytoplasmic red dye. Sinusoidal stenosis model group, HE staining of liver cell volume increases, the cytoplasm stained light, some bright, large lipid droplets in the cell memory, manifested as severe diffuse fatty change. ShuangHua granules of high, medium and low dose group liver cells arranged in cords, no significant narrow sinus, light staining cytoplasm, but the smaller intracellular lipid droplets, the majority of liver steatosis to a lesser extent, byχ2 test, ShuangHua granules 1.9 g·kg-1 and lovastatin groups had significantly different compared with model group (P< 0.05).11. The effect of Shuanghua granules on LDLR, PPARy protein expression in hepatic cells of prey hyperlipidemia rats Compared with normal rats, the hepatic LDLR, PPARy protein expression of model group were significantly decreased(P<0.01). ShuangHua granules could significantly promote the hepatic LDLR, PPARy protein expression, compared with the model group, there was significant differences (P<0.05, P<0.01).Conclusion:ShuangHua granules can effectively prevent acute, exogenous and prey hyperlipidemia. ShuangHua granules could improve antioxidant capacity, inhibit lipid peroxide production and correct abnormal blood rheology caused by hyperlipidemia, it also could increase LPL, HL, LA activity. There had no significant changes on fecal lipid excretion. The lipid regulating mechanism of ShuangHua granules is not increasing lipid excretion, it may be related to the lipid peroxidation, improve blood rheology, increase LPL, LA, HL activity. The possible mechanism of molecular biology is obviously increasing the protein expression of PPARy and LDLR in hepatic cells of rats. |
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