Mechanism of puerarin reversing SH-SY5Y cell injury induced by Aβ_(1-42) based on proteomics / 中国中药杂志

Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ，the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.

Effect of Xiao Chaihutang on NF-κB Signaling Pathway in Synovial Tissue of CFA Rats / 中国实验方剂学杂志

Objective:To observe the expression of tumor necrosis factor receptor-associated death domain (TARDD), nuclear transcription factor-κB inhibiting protein α(IκBα)IκB kinase-α (IKKα) and nuclear transcription factor (NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant (CFA) rats after treatment with Xiao Chaihutang (XCHT). Method:In animal experiments, SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table, the rats were randomly divided into the normal group, the model group, the low-dose XCHT group, the medium-dose XCHT group, the high-dose XCHT group, and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5.94 g·kg-1),medium-dose XCHT group(11.88 g·kg-1),high-dose XCHT group(23.76 g·kg-1),Tripterygium glucosides group(0.006 3 g·kg-1) were given corresponding drugs by gavage for three times a day, 2 mL/time. The histopathology of rat ankle joint was observed, and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot. Result:With the increase of the dosage of XCHT, the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats, compared with the model group, the statistical results of the low-dose XCHT group showed decreased protein expressions (PPPα, IκB α, NF-κB p65 in the NF-κB signaling pathway were significantly increased (PPα, IκBα, NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower (PPConclusion:This study shows that as the dose of Xiao Chaihutang increases, it could effectively improve synovitis, and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB, thereby preventing inflammation and suppressing bone erosion.

Molecular mechanism of Erchen Decoction in treatment of coronary heart disease based on network pharmacology / 中草药

Objective To explore the targets, metabolites, and signal pathways of Erchen Decoction based on network pharmacology and to reveal the molecular mechanism of Erchen Decoction in the treatment of coronary heart disease (CHD) by “phlegm and blood stasis”. Methods TCMSP, TCM-PTD, and TCMID were used to find the main ingredients of Erchen Decoction. Targets of Erchen Decoction were predicted by using ChemMapper database. The targets related with coronary heart disease were obtained by searching through TTD, Drugbank, and DisGeNET databases. The common targets of disease-drug component were selected by intersecting disease targets with drug targets. A protein-protein interaction (PPI) network model was constructed using the STRING platform. The metabolites of above-mentioned and selected components were analyzed using the MetaboAnalyst database to screen out the main metabolites. MetScape and ClueGO APP in Cytoscape were used to analyze the gene function and metabolic pathway of Erchen Decoction in the treatment of CHD, and to construct the main drug component-metabolite-target-signal pathway topology network of Erchen Decoction. Results Eight main drug ingredients of Erchen Decoction, 56 predicted targets related with CHD, and 13 metabolites and 13 signal pathways related to the main components of Erchen Decoction were screened out by network analysis at the same time. The molecular network of compounds of Erchen Decoction-metabolites-CHD targets-signal pathways were constructed based on all the targets. The results showed that Erchen Decoction may play a therapeutic role in CHD by protecting vascular endothelium, anti-oxidative stress, anti-inflammatory and injury repair by multi-targets and multi-pathways. Conclusion This research explores the possible molecular mechanism of Erchen Decoction in the treatment of CHD by “phlegm and blood stasis” and reveals the pharmacological effects of Erchen Decoction to provide direction for carrying out the follow-up evaluation of curative effect of Erchen Decoction in clinic.

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.

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.

Active part of Achyranthes promotes maturation and regulates the functions of dc in tumor microenvironment / 中国药学杂志

OBJECTIVE: To study the active part of Achyranthes bidentata promotes maturation and regulates the functions of DC in tumor microenvironment. METHODS: The total saponins and the polysaccharides from dry powder of Achyranthes bidentata were extracted and identified. The tumor microenvironment by co-culturing breast cancer MCF-7 cells with DCs, and add polysaccharides, total saponins or total saponins with polysaccharides of Achyranthes bidentata to stimulate DCs were established. The expression of surface molecules on DCs and the killing activity of cytotoxic T lymphocytes (CTL) induced by DCs by Flow Cytometry were detected. ELISA was used to detect the levels of IL-β, TNF-α, IL-10 and IL-12 in the culture supernatant. Detect the protein expression of JAK, p-JAK, STAT and p-STAT3 in DCs by Western blot. RESULTS: In the tumor microenvironment, the active parts of Achyranthes bidentata, total saponins and ABPS, alone or combined used, can trigger JAK/STAT3 signaling pathway of DCs through TLR4 and Dectin-1 receptor and upregulate the expression of surface molecules, such as CD80, CD86 and CD40, to induce the maturation and differentiation of DCs, promote the secretion of TNF-α, IL-12, IL-10 and IL-β. CONCLUSION: As an effective adjuvant, the active parts of traditional Chinese medicine can enhance the effect of DC vaccine to correct the immune deficiency in tumor microenvironment, activate tumor-specific CTLs and then produce specific anti-tumor effects.

Effect of ASO Blood Stasis Syndrome Serum on Vascular Endothelial Cell Injury and Regulation of Taohong Siwu Decoction on it / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of arteriosclerosis obliterans (ASO) blood stasis syndrome (BSS) serum on vascular endothelial cell injury and to study the regulation of Taohong Siwu Decoction (TSD) on it.</p><p><b>METHODS</b>Umbilical vein endothelial cell culture system was established. The serum endothelial cell injury model with ASO BSS was prepared. Low, medium, and high concentrations TSD containing serums were respectively added. The endothelial cell proliferation activity was observed by MTT method. Ultrastructures of endothelial cells were observed under transmission electron microscope. Changes of intracellular calcium ion concentration and the cytoskeleton were observed under laser confocal microscope. Contents of ET, NO, and transforming growth factor beta1 (TGF-beta1) in endothelial cell culture supernatant were detected by ELISA.</p><p><b>RESULTS</b>In ASO BSS serum group endothelial cell proliferation activities decreased, the cell structure was obviously destroyed, calcium ion concentration increased, contents of ET, NO and TGF-beta1 increased significantly (P < 0.01), and ET/NO ratio was imbalanced. After incubating with TSD drug containing serum, endothelial cell proliferation activities and injured cell structures were obviously improved; ET, NO and TGF-beta1 levels decreased (P < 0.05, P < 0.01), ET/NO ratios approximated to the normal level.</p><p><b>CONCLUSION</b>The main mechanism of TSD for treating ASO ASS lied in improving injured vascular endothelial cells and endocrine disorder.</p>

Taohong Siwu Decoction regulated functions of endothelial cells and treated arteriosclerosis obliterans: an experimental study / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To discuss the effect of Taohong Siwu Decoction (TSD) in regulating functions of endothelial cells and treating arteriosclerosis obliterans (ASO).</p><p><b>METHODS</b>The ASO model was prepared by using high-fat diet plus intimal injury. They were randomly divided into the model group (n = 10), the normal control group (n = 9), the low dose TSD group (group A, n = 12), the middle dose TSD group (group B, n = 10), and the high dose TSD group (group C, n = 9). Eight weeks after modeling, the limb blood perfusion was observed using laser Doppler flowmetry. The arterial morphology was observed using light microscope and transmission electron microscope. The number of circulating endothelial cells (CECs) was determined using Percoll density gradient centrifugation method. Serum levels of TNF-alpha, IL-1, ET-1, and NO were detected using double antibody sandwich assay of enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The ASO rat model was successfully established. Blood lipids levels significantly increased, the blood perfusion of left hind limbs significantly decreased, the number of CECs in the peripheral blood significantly increased, the arterial lumen was irregularly narrowed, the ultra-structure of vessel walls was damaged, serum levels of TNF-alpha, IL-1, and ET-1 significantly increased, and the serum level of NO significantly decreased in the model group, showing statistical difference when compared with the normal control group (P < 0.01). Compared with the model group, significant improvement in the aforesaid indices was shown in group B and C (P < 0.05, P < 0.01).</p><p><b>CONCLUSIONS</b>The injury and abnormal functions of endothelial cells is an important pathological process of ASO. As an effective recipe for treating ASO, TSD could protect vascular endothelial cells and improve the secretion function of vascular endothelial cells.</p>

Signal transduction mechanism of macrophages polarization induced by methionine enkephalin / 中国药学杂志

OBJECTIVE: To investigate the signal transduction mechanism of macrophages polarization induced by methionine enkephalin (MENK), which can promote tumoricidal responses in vitro. METHODS: The phenotype of macrophages were assessed by the quantitative analysis of key surface molecules on macrophages with flow cytometry (CD64, CD206). The expressions of NF-kB/STAT6 signal transduction were analyzed with Western-blotting. RESULTS: MENK(10-12 mol · L-1) could significantly decrease the expression of CD206 (P < 0.01), and at the same time, it could increase the expression of CD64 significantly (P < 0.01). MENK could increase the expression of NF-κB compared with MENK-untreated group. Furthermore MENK could inhibit the activation of STAT6 which is mediated by IL4 (P < 0.05). CONCLUSION: MENK could inhibit the activation of STAT6 signaling pathway mediated by IL4. Meanwhile, MENK could increase the expression of NF-κB and be conducive to the combination of NF-kB sites with M1-type cytokines, consequently MENK could induced the conversion of macrophages from M2 to M1 phenotype effectively.