Effect and Mechanism of Xiangsha Liujunzi Tang on Lipid Deposition in Liver of ApoE-/- AS Mice by Affecting Long Noncoding RNA-HC/miR-130b and to Regulate Cholesterol Metabolism / 中国实验方剂学杂志

Objective:To investigate the mechanism of Xiangsha Liujunzi Tang in improving liver lipid deposition in ApoE^-/- atherosclerotic （AS） mice by affecting long noncoding RNA-HC （Lnc-HC）/microRNA-130b （miR-130b） in the regulation of cholesterol metabolism. Method:Totolly 10 C57BL/6J mice were selected as normal controls， and 30 healthy ApoE^-/- mice fed with high fat diet for 12 weeks were then randomly divided into the model group， Xiangsha Liujunzi Tang group（19.12 g·kg^-1·d^-1） and simvastatin group（2.275 mg·kg^-1·d^-1）， with gavage administration for 4 weeks. The serum lipid level of mice was detected by automatic biochemistry analyzer， and the histopathological changes of liver cells were observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect expression of long noncoding RNA-HC， and miR-130b. Real-time PCR and Western blot assay were used to detect gene and protein expression of peroxisome proliferator-activated receptor gamma （PPAR<italic>γ</italic>）， liver X receptor （LXR）， ATP-binding cassette transporters A1 （ABCA1）， ATP-binding cassette transporters G1 （ABCG1）， ATP-binding cassette transporters G5 （ABCG5）， and ATP-binding cassette transporters G8 （ABCG8）. Result:Compared with the normal control group， the mice in the model group showed abnormal blood lipids， larger liver cells， obvious fat vacuoles， significantly increased expression of Lnc-HC， miR-130b in liver， and significantly decreased gene and protein expression of PPAR<italic>γ</italic>， LXR， ABCA1， ABCG1， ABCG5， and ABCG8 in mice liver （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the model group， the abnormal blood lipid levels of the mice in the Xiangsha Liujunzi Tang group and the simvastatin group were improved， and the number of fatty vacuoles of liver cells was significantly reduced， the expression of liver Lnc-HC， miR-130b in Xiangsha Liujunzi Tang group decreased significantly （<italic>P</italic><0.05，<italic>P</italic><0.01）， the gene and protein levels of liver PPAR<italic>γ</italic>， ABCA1， ABCG1， ABCG5， ABCG8 in mice of the Xiangsha Liujunzi Tang group and the simvastatin group showed an upward trend. Among them， the gene and protein expression of LXR protein in the liver of the Xiangsha Liujunzi Tang group was significantly up-regulated （<italic>P</italic><0.05）. Conclusion:Xiangsha Liujunzi Tang may improve the lipid deposition in the liver of ApoE^-/-AS mice by affecting Lnc-HC/miR-130b to regulate the cholesterol metabolism process mediated by PPAR<italic>γ</italic>， thus playing a role in preventing and treating AS.

Study on Inhibitory Effect of Huayu Qutan Recipe on Myocardial Fibrosis in Atherosclerotic Rabbits Based on Mitochondrial Fusion-lysis / 中国实验方剂学杂志

Objective::To analysis the effect of Huayu Qutan recipe on myocardial fibrosis in atherosclerotic rabbits based on mitochondrial fusion-lysis. Method::The 36 SPF healthy male rabbits were selected, and 6 rabbits were selected randomly as the normal group, and given normal pellet feed, another 30 rats were fed with high fat diet to establish atherosclerosis model.After successful replication of animal models, they were randomly divided into model group, Huayu Qutan recipe low-dose, medium-dose and high-dose group (4.0, 8.0, 16.0 g·kg-1) and simvastatin group (1.4 mg·kg-1), 6 rats each.Each group was given corresponding drugs according to the dosage, continuous administration for 4 weeks.The serum lipid levels in rabbits of each group were detectived by automatic biochemical analyzer, the degree of myocardial fibrosis was measured by Masson staining, and expression levels of mitochondrial fusion protein 1 (Mitofusin 1), mitochondrial fusion protein 2 (Mitofusin 2), optic atrophy protein 1 (Opa1), promoter protein 1 (Drp1), mitogen 1 (Fis1) in myocardial tissue were detected by immunohistochemistry. Result::Compared with normal group, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) in model group were significantly increased, levels of density lipoprotein cholesterol (HDL-C) were significantly decreased, the expression levels of Mitofusin 1, Mitofusin 2 and Opa1 in myocardial tissue were significantly decreased, the expression levels of Drp1 and Fis1 were significantly increased(P<0.05, P<0.01), compared with model group, the levels of serum TC, TG, LDL-C in simvastatin group and Huayu Qutan recipe low-dose, medium and high-dose group were significantly decreased, levels of HDL-C were significantly increased, the expression levels of Mitofusin 1, Mitofusin 2 and Opa1 in myocardial tissue were significantly increased, the expression levels of Drp1 and Fis1 were significantly decreased (P<0.05, P<0.01), compared with the Huayu Qutan recipe high-dose group, the levels of serum TC, TG, LDL-C in simvastatin group and Huayu Qutan recipe low, medium-dose group were significantly increased, the expression levels of Mitofusin 1, Mitofusin 2 and Opa1 in myocardial tissue were significantly decreased, the expression levels of Drp1 and Fis1 were significantly increased(P<0.05, P<0.01). Conclusion::Huayu Qutan recipe can effectively regulate blood lipid and inhibit myocardial fibrosis in atherosclerotic rabbits, and the higher the dose of Huayu Qutan recipe, the more obvious the effect is, it is speculated that its effect may be related to the regulation of the expression of mitochondrial fusion-lysis related proteins such as Mitofusin1, Mitofusin2 and Opa1, Drp1 and Fis1 in myocardial cells.

Effect of Xiangsha Liu Junzitang on dyHDL in Hyperlipidemia Rats with Spleen Deficiency / 中国实验方剂学杂志

Objective: To observe the changes of dysfunctional high density lipoprotein cholesterol (dyHDL) and the intervention effect of Xiangsha Liu Junzitang in rats with spleen deficiency and hyperlipidemia, and reveal the effect and mechanism of Xiangsha Liu Junzitang on dyHDL in rats with spleen-deficiency hyperlipidemia. Method: Seventy-five SPF SD rats were randomly divided into normal group, high fat group, spleen deficiency and high fat group, Xiangsha Liu Junzitang low and high dose groups (5.67, 11.34 g·kg-1). In the spleen deficiency and high fat group, as well as Xiangsha Liu Junzitang low and high dose groups, composite method of improper diet and exhaustive swimming was used for 15 days for modeling. After modeling for 15 days, normal group was fed with basic diet, while the high-fat group, spleen-deficiency and high-fat group, the Xiangsha Liu Junzitang low and high dose groups were fed with high-fat diet. After 12 weeks, the Xiangsha Liu Junzitang low dose and high dose groups received corresponding dosage of drugs, while normal group, high fat group and spleen deficiency high fat group received corresponding volume of normal saline. After 4 weeks, the contents of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol cholesterol (LDL-C), and high density lipoprotein cholesterol(HDL-C)were detected by automatic biochemistry analyzer, while D-xylose excretion rate was measured by phloroglucinol method. The morphological changes of liver cells were observed by hematoxylin eosin (HE) staining. The level of PON1, apoA1 and SAA in plasma were detected by enzyme-linked immunosorbent assay (ELISA). Paraoxonase 1(PON1), apolipoprotein A1 (apoA1) and serum amyloid protein A (SAA) gene expression in rats liver were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result: As compared with normal group, the serum TC, TG, and LDL-C levels were significantly increased in the high-fat group and spleen-deficiency high-fat group (PPPPPPD-xylose excretion rate was significantly decreased in the spleen-deficiency and high-fat group (PPPPPPPPConclusion: The lipid disorder in hyperlipidemia rats was aggravated by the spleen deficiency, but was corrected after intervention with Xiangsha Liu Junzitang. and its mechanism may be related to the regulation of the expression of dyHDL-related genes and protein.

Study on Serum Metabolite Profiling in Pi-deficiency Rats Based on LC-MS Technique / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore metabolite profiling changes in serum of rats with pi-qi deficiency syndrome (PQDS) and pi-yang deficiency syndrome (PYDS) based on liquid chromatograph-mass spectrometer (LC-MS) technique, and to explore the essence of Pi-deficiency syndrome (PDS) from small molecule metabolite level.</p><p><b>METHODS</b>Totally 21 male SD rats of SPF grade were randomly divided into three groups, the normal control group, the PQDS group, and the PYDS group, 7 in each group. Rats in the PQDS group overate for 1 day and fasted for 2 days. They drank freely and then swam to be exhausted in water at 35 degrees C - 37 degrees C for 15 successive days. The PYDS model was established by the same method for PQDS rats plus drenching 20% Folium sennae water extract (2 mL/100 g), once in the morning and once in the evening for one successive week. After modeling, models were evaluated according to rat general state, changes in body weight and rectal temperature. Serum metabonomic profiles were detected using LC-MS technique. Difference in inter-group metabolite spectrograms was analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA). Potential biomarkers related to syndrome types in rat serum were selected via the parameter of variable importance in the projection (VIP).</p><p><b>RESULTS</b>The weight of rats in the PQDS group and the PYDS group decreased more significantly after modeling. The difference in prepost weight was statistically significant from that of the normal control group (P < 0.01). It was more obviously lowered in the PYDS group than in the PQDS group (P < 0.05). Compared with the normal control group, the rectal temperature of rats in the PYDS group and the PQDS group decreased (P < 0.05, P < 0.01). It decresed more in the PYDS group than in the PQDS group (P < 0.05, P < 0.01). Compared with the normal control group, levels of PC(19:0)/PE(22:0), PC(17:0)/PE(20:0), capric acid, oleic acid, stearic acid, succinic acid, fumaric acid, malic acid, glucose increased; arachidonic acid, linolenic acid, lauric acid, androsterone, 4-heptanone, dihydroxyacetonephosphate (DHAP) (6:0), and uridine decreased in the PYDS group and the PQDS group. Compared with the PQDS group, levels of PC(22:1), PC (22:6), PE (18:0)/PC (15:0), retinol, and deoxycytidine increased significantly in the PYDS group; PC (18:1), PC(19 :3), PC (20:3), PC (17:0)/PE (20:0), PC (19:1)/PE (22:1), PC (19:0)/PE (22:0), PC (17:1)/PE (20: 1), PC (16:1)/PE (19:1), cholic acid, hippuric acid, furoic acid, undecanedicarboxylic acid, palmitoleic acid, hydroxy stearic acid, eicosatrienoic acid, phenylalanine, tyrosine, glutamic acid, serine, carbamoyl aspartic acid, palmitoyl carnitine, tetradecanoyl carnitine, acetylcarnitine, and linoleylcarnitine decreased more significantly in the PYDS group.</p><p><b>CONCLUSIONS</b>Comparative contents of various serum metabolites changed significantly in PQDS and PYQS model groups. Some potential small molecular biomarkers related to PDS were preliminarily identified. These results might provide some data reference for exploring scientific connotation and pathological mechanisms of PDS.</p>

Correlation between autophagy and polarization of macrophages in atherosclerosis plaque in arteriosclerosis obliterans amputees / 药学学报

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor- α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P < 0.01 or 0.05), and arginase-1 (Arg-1), transforming growth factor-β (TGF-β), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P < 0.01). The levels of p-STAT1 and NF-κB were significantly increased in fibrous plaque (P < 0.01), while p-STAT6 expression was significantly increased in atheromatous plaque (P < 0.01). The level of LC3-II was significantly higher in atheromatous plaque than that in fibrous plaque (P < 0.01). Macrophages in early atherosclerotic plaque were induced to M1 type through p-STAT1/NF-κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.

Correlation between autophagy and polarization of macrophages in atherosclerosis plaque in arteriosclerosis obliterans amputees / 药学学报

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P β (TGF-β), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P κB were significantly increased in fibrous plaque (P P P κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.

Intervention of Huayu Qutan Recipe on liver SREBP-2 signal pathway of hyperlipidemia rats of pi deficiency syndrome / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the intervention of Huayu Qutan Recipe (HQR) on liver SREBP-2 signal pathway of hyperlipidemia rats of Pi deficiency syndrome (PDS).</p><p><b>METHODS</b>Totally 100 SPF grade SD rats were randomly divided into the blank control group, the hyperlipidemia group, the hyperlipidemia treatment group, the PDS hyperlipidemia group, and the PDS hyperlipidemia treatment group, 20 in each group. Common granular forage was fed to rats in the blank control group. High fat forage was fed to rats in the hyperlipidemia group and the hyperlipidemia treatment group. Rats in the PDS hyperlipidemia group and the PDS hyperlipidemia treatment group were treated with excessive labor and improper diet for modeling. They were administered refined lard by gastrogavage (3 mL each time, twice per day) and fed with high fat forage on the odd days, and fed with wild cabbage freely on even days. The modeling lasted for 30 days. Rats in the hyperlipidemia treatment group and PDS hyperlipidemia treatment group were administered with Huayu Qutan Recipe (20 mL/kg) by gastrogavage, once a day, for 30 successive days. Levels of serum cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and serum amylase (AMY) were detected by automatic biochemical analyzer. D-xylose excretion rate was determined using phloroglucinol method. Morphological changes of liver and the lipid deposition in liver were observed using HE stain and oil red O stain respectively, mRNA and protein expression levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol 7α-hydroxylase 1 (CYP7A1), LDL-R, and sterol regulatory element binding protein-2 (SREBP-2) were detected using real time RT-PCR and Western blotting.</p><p><b>RESULTS</b>Compared with the blank control group, serum levels of TC (1.84 ± 0.19 mmol/L, 2.23 ± 0.43 mmol/L) and LDL-C (0.99 ± 0.24 mmol/L, 1.13 ± 0.56 mmol/L) were higher in the hyperlipidemia group and the PDS hyperlipidemia group, serum levels of HDL-C (0.41 ± 0.66 mmol/L, 0.41 ± 0.11 mmol/L) and AMY activities (351 ± 45 mmol/L, 153 ± 30 mmol/L) were lower, and urinary D-xylose excretion rates were lower (26.9 ± 2.1 ng/mL, 15.0 ± 1.7 ng/mL) (all P < 0.05). Lipid deposition occurred in liver cells. Much fat vacuoles occurred in the cytoplasm. Expression levels of HMGCR, CYP7A1, LDL-R, and SREBP-2 mRNA and proteins in liver significantly decreased (P < 0.01). Compared with the hyperlipidemia group, serum levels of TC and LDL-C significantly increased (P < 0. 05), AMY activities and urinary D-xylose excre- tion rates significantly decreased in the PDS hyperlipidemia group (P < 0.01). A large amount of lipid deposition occurred in liver. The atrophy of liver cells was obviously seen. Expression levels of CYP7A1, LDL-R, and SREBP-2 mRNA and proteins in liver were significantly lower (P < 0.01, P < 0.05). Serum levels of TC and LDL-C significantly decreased (P < 0.05), AMY activities and urinary D-xylose excretion rates significantly increased in the hyperlipidemia treatment group (P < 0.01). Expression levels of CYP7A1, LDL-R, and SREBP-2 mRNA and proteins in liver were significantly increased (P < 0.01, P < 0.05). Compared with the PDS hyperlipidemia group, serum level of TC significantly decreased (P < 0.05), HDL-C levels, AMY activities and urinary D-xylose excretion rates significantly increased in the PDS hyperlipidemia treatment group (P < 0.01),expression levels of CYP7A1, LDL-R, and SREBP-2 mRNA and proteins in liver were significantly increased (P < 0.01). Similar changes occurred in the two treatment groups.</p><p><b>CONCLUSIONS</b>Pi deficiency exacerbates abnormal serum TC level and the lipid deposition in liver. These might be related to regulating expression levels of LDL-R, HMGCR, and CYP7A1 genes in the SREBP-2 signal pathway. HQR could regulate this pathway to intervene abnormal metabolism of TC.</p>

Comparative proteomics analysis of the ileum of Pi-Yang deficiency model rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>Based on proteomics technology, Pi-yang deficiency syndrome (PYDS) correlated differential proteins were screened, thus providing powerful experiment reliance for exploring the essence of PYDS.</p><p><b>METHODS</b>Totally 36 SD rats of SPF grade were randomly divided into the normal control group (n = 16) and the PYDS group (n = 20). The PYDS model rats were induced by improper diet, overstrain, and administration of yang impairing bitter cold herbs. The total proteins of the ileum were separated and extracted from rats in the PYDS group and the normal control group. The differential protein dots were identified using Delyder 2D 6.5 image analysis software by two-dimensional gel electrophoresis (2-DE) technology. The finger print map of corresponding peptide qualities was obtained by applying MALDI TOF/TOF. The differential proteins were identified using Mascot search library.</p><p><b>RESULTS</b>Judged by statistics and fuzzy mathematics, Pi-yang deficiency rat model was successfully established. Eight proteins with differential expressions involving cell skeleton, energy metabolism, and signal transduction, and so on were obtained. Of them, there were 4 up-regulated proteins, i.e., desmin, cytokeratin8 (CK8), pyruvate kinase (PK), and ezrin. Four down-regulated proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytokeratin19 (CK19), cytokeratin1 (CK1), and actin.</p><p><b>CONCLUSION</b>The pathogenesis of PYDS might be slowed energy metabolism rate, reduced energy production, changed structure of ileal villin, and weakened absorbing and digestive functions.</p>

Experimental study on the ileum tissue proteins differentially expressed in Pi-qi deficiency syndrome rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To identify the ileum tissue proteins differentially expressed in Pi-qi deficiency syndrome rats using proteomic approach.</p><p><b>METHODS</b>Thirty-seven rats were randomly divided into 3 groups, i. e., the normal control group (Group 1), the Pi-qi deficiency syndrome model group (Group 2), and the reserpine group (Group 3). The Pi-qi deficiency syndrome model was established using excessive exerting combined with irregular diet, and peritoneally injecting Reserpine Injection (1 mg/mL) respectively. The ileum tissues were separated after identified fuzzy method. The differentially expressed proteins were separated with two dimensional electrophoresis (2-DE), analyzed by Mass Spectrometry, and identified by MASCOT Software.</p><p><b>RESULTS</b>Three proteins were differentially expressed in Group 2 and nine proteins were differentially expressed in Group 3 (P < 0.05).</p><p><b>CONCLUSION</b>Pi-qi deficiency syndrome was closely related with decreased expression of albumin as well as increased expressions of trypsin and glucose-regulated protein 78.</p>