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Objective:To explore the improvement effect of total flavonoids of Mori Cortex combined with total saponins of Anemarrhena Asphodeloide on hyperlipidemia rats with osteoporosis and its possible mechanism. Method:The 40 SPF male SD rats were adaptively fed for 7 days， and then randomly divided into normal group， model group， calcitriol group （45 ng·kg<sup>-1</sup>）， total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group （0.6 g·kg<sup>-1</sup>+0.4 g·kg<sup>-1</sup>） and 2∶1 group （1.2 g·kg<sup>-1</sup>+0.2 g·kg<sup>-1</sup>）. Except for the normal group， rats in the other groups were fed with high fat for 9 weeks， the normal group and the model grouotal flavonoids of total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloip were given normal saline by gavage， and the other groups were given corresponding drugs by gavage， after 12 weeks of administration， except for the normal group ， the other groups were given intramuscular injection of glucocorticoids at the same time. After 22 weeks of administration， the weight of rats with total flavonoids from Mori Cortex combined with total saponins of Anemarrhena Asphodeloide was measured. Serum levels of total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C）， osteocalcin （BGP） and bone alkaline phosphatase （BALP） were determined by biochemical assay. Hematoxylin-eosin （HE） staining to observe the pathological changes of rat tibia. Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） was used to detect the expression levels of peroxisomal proliferators activate the receptor gamma（PPAR<italic>γ</italic>） and Runt-related transcription factor 2 （Runx2） mRNA in rat bone tissue， immunofluorescence was used to detect the expression of PPAR<italic>γ</italic> and Runx2 in rats. Result:Compared with normal group， the body mass of rats in model group was significantly increased （<italic>P</italic><0.01）， and the contents of TC， TG， and LDL-C in the serum were significantly increased （<italic>P</italic><0.01）. Compared with model group， the body weight of rats in thet total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group were significantly reduced （<italic>P</italic><0.01）， and the contents of TC， TG， and LDL-C in the serum were significantly reduced （<italic>P</italic><0.01）， the content of BGP and BALP increased （<italic>P</italic><0.01）. HE staining results showed that compared with the normal group， the tibia fat vacuoles of the model group increased， and the number of osteoblasts decreased， compared with the model group， the total flavonoids of the Mori Cortex and the flavonoids-total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group decreased in tibia fat vacuoles and increased the number of osteoblasts， the results of immunofluorescence and Real-time PCR showed that， compared with normal group， the expression of Runx2 in the model group decreased and the expression of PPAR<italic>γ</italic> increased （<italic>P</italic><0.01）. Compared with model group， the total flavonoids of Mori Cortex-total saponins 1∶2 group and the total flavonoids of Mori Cortex-total saponins 2∶1 Group up-regulated the expression of Runx2 and down-regulated the expression of PPAR<italic>γ </italic>（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:The total flavonoids of Mori Cortex combined with the total saponins of Anemarrhena Asphodeloide up-regulated Runx2 and down-regulated the expression of PPAR<italic>γ</italic> mRNA and protein， thereby affecting the metabolism of TG and TC in the blood， achieving a therapeutic effect on osteoporosis， provides experimental basis for the clinical prevention and treatment of hyperlipidemia with osteoporosis.
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Objective::To observe the effect of sanggenone C (SanC) on the proliferation and differentiation of mouse MC3T3-E1 osteoblasts induced by dexamethasone (DEX), and to explore its mechanism. Method::Molecular docking was conducted between SanC and Runt-associated transcription factor 2(Runx2) protein structure obtained by homologous modeling. MC3T3-E1 cells were jointly treated by different concentrations of SanC (8, 16, and 32 μmol·L-1) and 1 μmol·L-1 DEX, and then cell counting kit-8(CCK-8) method was used to detect the effect of SanC on the proliferation of MC3T3-E1 osteoblasts. The alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts was determined by reagent kit and the formation of mineralized bone nodules were detected by alizarin red staining. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of Runx2, ALP and Osterix. The protein expression of Runx2 was detected by Western blot. Result::The docking score of SanC and Runx2 was -9.78.As compared with the normal group, DEX group significantly reduced the cell survival rate (P<0.01), and the greatest difference occurred on the seventh day. As compared with DEX group, SanC could significantly promote the cell proliferation of MC3T3-E1 (P<0.01), in which 32 μmol·L-1 SanC had the largest difference in proliferation rate on seventh day. As compared with the normal group, the expression of Runx2, ALP and Osterix mRNA increased to a certain extent in DEX group(P<0.01). As compared with DEX group, the expression levels of Runx2, ALP and Osterix mRNA were up-regulated in different concentration groups of SanC in a dose-dependent manner (P<0.01). As compared with the normal group, the expression of Runx2 protein in DEX group decreased significantly (P<0.05), and as compared with DEX group, the expression of Runx2 protein in cells under the intervention of SanC increased significantly (P<0.01). Conclusion::SanC can promote the proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, and the mechanism may be related to the up-regulation of Runx2 expression.
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Objective:To explore the effect of anemarrhena asphodeloside BⅡ (TBⅡ) on the expressions of nuclear transcription factor-κB receptor activator factor ligand （RANKL）, RANK and C-FOS genes during osteoclast differentiation. Method:Molecular docking software LeDock was used to score the docking of TBⅡ with RANKL, RANK and C-FOS. RAW264.7 was treated with soluble RANKL（sRANKL） and divided into control group, sRANKL group (model group), Icariin (Ica) group, low-dose TBIⅡ group (2 μmol·L-1), medium-dose TBⅡ group (4 μmol·L-1), and high-dose TBⅡ group (8 μmol·L-1). The corresponding kit was used to detect iconic enzyme (TRAP) of osteoclast differentiation. Total RNA was extracted by trizol method, Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expressions of C-FOS, upstream RANKL/RANK and downstream nuclear factor of activated T-cells cytoplasmic 1 (NFATC1), and osteoprotegerin OPG. Result:The molecular docking score were -11.86, -11.38, -12.34 kcal·mol-1, and there might be multiple binding sites between TBII as well as RANKL, RANK and C-FOS. Compared with the control group, the content of TRAP in model group increased significantly (P<0.01), and compared with model group, the content of TRAP in each administration group decreased significantly (P<0.01), and TBⅡ decreased the content of TRAP in a dose-dependent manner. Compared with the control group, the expressions of RANKL, RANK, C-FOS and NFATC1 increased (P<0.01), whereas the expression of OPG decreased (P<0.01) in model group. Compared with model group, the expressions of RANKL, RANK, C-FOS and NFATC1 decreased (P<0.01), while the expression of OPG increased (P<0.01) in each administration group. Conclusion:TBⅡ may inhibit the differentiation of osteoclast precursors into osteoclasts, inhibit osteoclast activity, reduce bone resorption and improve osteoporosis by regulating RANKL/RANK/C-FOS signal pathway.
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Objective To investigate the mechanism of total flavonoids of Mori Cortex (TFMC) in improving hyperlipidemia and hyperuricemia related nephropathy. Methods The molecular structure of URAT1 protein and structure of Mori Cortex total flavonoids extract were docked by selecting the effective components of total flavonoids extract of Mori Cortex and related genes of uric acid. Forty SD rats were randomly divided into four groups: normal group, model group, TFMC group (1.0 g/kg) and benzbromarone group (6.25 mg/kg). The hyperlipidemia and hyperuricemia model was established by feeding with high fat diet plus adenine and ethylamine butanol. After 16 weeks, the levels of blood glucose and blood lipids in serum, uric acid (UA), creatinine (CRE), and urea nitrogen (BUN) of rats in each group were compared. The renal pathological changes were observed by hematoxylin eosin staining. The expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), uric acid anion exchange transporter 1 (URAT1) and organic anion transporter 1 (OAT1) were detected by qRT-PCR. Results The main component of moracin of total flavonoids of Mori Cortex was the high target of the genes related to uric acid, suggesting that the moracin might be the main active component in the improvement of hyperlipidemia and hyperuricemia related nephropathy. After 16 weeks of drug intervention, the serum levels of Glu, TC, TG, LDL-C, UA, CRE and BUN in the model group were significantly higher than those in the normal group, and the level of HDL-C in the model group was significantly higher than that in the normal group (P < 0.05, 0.01). Compared with the model group, the above biochemical indexes in the TFMC group and the benzbromarone group were significantly decreased and HDL-C was significantly increased (P < 0.05, 0.01). The results of HE staining showed that the epithelial cells of renal tubules in the model group were swollen and necrotized, and the protein tubules could be seen in the renal tubules. In the TFMC group, some renal tubular epithelial cells were slightly swollen, and no inflammatory cells infiltrated around them. The structures of renal cortex and medullary were clear, and no hyperplasia or atrophy in glomerular were found, no tubular type and inflammatory cell infiltration in renal interstitial tissue of rats in benzbrommarone group were observed. The results of qRT-PCR showed that, compared with the normal group, the content of IL-6, TNF-α, and URAT1 mRNA was significantly increased, the content of OAT1 mRNA was significantly decreased in the model group; The content of above indicators was decreased and OAT1 was increased after drug intervention, (P < 0.05, 0.01). Conclusion The improvement of hyperlipidemia and hyperuricemia associated nephropathy may be related to the regulation of IL-6, TNF-α, URAT1, and OAT1 mRNA. Mori Cortex has obvious influence on the key factor of hyperlipidemia and hyperuricemia with URAT1 as its potential target, and the results of molecular virtual docking and animal experiments are similar. It provides a theoretical basis for further study on the improvement of hyperlipidemia and hyperuricemia related nephropathy and provides a reference for the further molecular mechanism.
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To investigate the mechanism of the treatment of hyperlipidemia rats induced by Huangqi San. The 40 male SD rats were randomly divided into normal group, model group, Huangqi San low and high dose group (1, 2 g·kg⁻¹), and positive lipitor group (2 mg·kg⁻¹). The normal group feeds on base feed, and other groups feed on high-fat feed. After 8 weeks, the hyperlipidemia model was successful. After intervention by drugs for 13 weeks, fasting blood glucose, total cholesterol, triglycerides and LDL cholesterol content of all rats were measured. The pathological changes of liver and skeletal muscle of rats were observed in rats. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of AMPK signaling pathway in the liver and skeletal muscles (AMPK, ACC, CPT1A, SREBP2, HMGCR). The degree of FPG, TC, TG and LDL-C were the highest in the model group, and the liver and skeletal muscle pathology were the most obvious. After intervention by Huangqi San and lipitor, a significant reduction in the blood sugar blood fat, liver, and skeletal muscle injury has improved significantly, except SREBF2 and HMGCR mRNA and protein expression of this enzyme is reduced, other AMPK pathway related mRNA and protein expression increased significantly. Huangqi San effect is superior to lipitor. Huangqi San may improve hyperlipidemia by regulating the AMPK signaling pathway, increasing the oxidation of fatty acids and inhibiting cholesterol synthesis.