[Objective]To explore the anti-cerebral ischemia mechanism of Rhubarb root and rhizome by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)combined with network pharmacology and in vitro experiment.[Methods]The active components of Rhubarb root and rhizome in rat serum samples were identified by UPLC-Q-TOF/MS.The potential targets of Rhubarb root and rhizome in blood components were obtained by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Swiss Target Prediction.The related targets of ischemic stroke(IS)were obtained from Genome Annotation Database Platform(GeneCards),Online Mendelian Inheritance in Man(OMIM)database and Therapeutic Target Database(TTD).The intersection targets were obtained by using Venny 2.1.0 software and imported into the STRING database for protein-protein interaction(PPI)network analysis.Cytoscape 3.7.2 software was employed to visualize and identify the core targets.Gene ontology(GO)enrichment function analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway enrichment analysis were conducted to predict the mechanism of action.Molecular docking was performed by AutoDock Vina.Finally,the network pharmacological prediction results were verified by in vitro experiments.[Results]A total of 15 active components were identified in serum containing Rhubarb root and rhizome(SR),and there were 87 intersection targets between Rhubarb root and rhizome and IS.KEGG enrichment analysis showed that Rhubarb root and rhizome may alleviate IS through phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)and nuclear factor-κB(NF-κB)signaling pathways.Molecular docking results showed that the core components had a strong binding ability to the key targets of Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/NF-κB signaling pathway.In vitro experiment demonstrated that SR reduced the production of reactive oxygen species(ROS)induced by oxygen-glucose deprivation/reoxygenation(OGD/R)in BV2 cells.It also increased the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),while decreasing the levels of malondialdehyde(MDA)and nitric oxide(NO).Additionally,SR reduced the levels of pro-inflammatory factors,increased the levels of anti-inflammatory factors in BV2 cells induced by OGD/R,and decreased the protein expression of TLR4,MyD88 and NF-κB in BV2 cells.[Conclusion]Rhubarb root and rhizome protects BV2 microglia from OGD/R-induced injury by inhibiting oxidative stress and inflammatory response,and its mechanism may be related to regulation of TLR4/MyD88/NF-κB signaling pathway.

[Objective]To explore the anti-cerebral ischemia mechanism of Rhubarb root and rhizome by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)combined with network pharmacology and in vitro experiment.[Methods]The active components of Rhubarb root and rhizome in rat serum samples were identified by UPLC-Q-TOF/MS.The potential targets of Rhubarb root and rhizome in blood components were obtained by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Swiss Target Prediction.The related targets of ischemic stroke(IS)were obtained from Genome Annotation Database Platform(GeneCards),Online Mendelian Inheritance in Man(OMIM)database and Therapeutic Target Database(TTD).The intersection targets were obtained by using Venny 2.1.0 software and imported into the STRING database for protein-protein interaction(PPI)network analysis.Cytoscape 3.7.2 software was employed to visualize and identify the core targets.Gene ontology(GO)enrichment function analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway enrichment analysis were conducted to predict the mechanism of action.Molecular docking was performed by AutoDock Vina.Finally,the network pharmacological prediction results were verified by in vitro experiments.[Results]A total of 15 active components were identified in serum containing Rhubarb root and rhizome(SR),and there were 87 intersection targets between Rhubarb root and rhizome and IS.KEGG enrichment analysis showed that Rhubarb root and rhizome may alleviate IS through phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)and nuclear factor-κB(NF-κB)signaling pathways.Molecular docking results showed that the core components had a strong binding ability to the key targets of Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/NF-κB signaling pathway.In vitro experiment demonstrated that SR reduced the production of reactive oxygen species(ROS)induced by oxygen-glucose deprivation/reoxygenation(OGD/R)in BV2 cells.It also increased the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),while decreasing the levels of malondialdehyde(MDA)and nitric oxide(NO).Additionally,SR reduced the levels of pro-inflammatory factors,increased the levels of anti-inflammatory factors in BV2 cells induced by OGD/R,and decreased the protein expression of TLR4,MyD88 and NF-κB in BV2 cells.[Conclusion]Rhubarb root and rhizome protects BV2 microglia from OGD/R-induced injury by inhibiting oxidative stress and inflammatory response,and its mechanism may be related to regulation of TLR4/MyD88/NF-κB signaling pathway.

Objective:To analyze the clinical characteristics, prognosis and gene mutation in patients with dilated cardiomyopathy (DCM) in Keshan disease area of Sichuan Province, and to explore the risk factors for all-cause death in DCM patients.Methods:In June 2016, 55 DCM patients diagnosed at the local disease prevention and control center through clinical manifestations, electrocardiogram examination, and echocardiography were selected as the survey subjects in Mianning County, Liangshan Yi Autonomous Prefecture, and Renhe District, Panzhihua City, Keshan disease areas of Sichuan Province. Baseline clinical data were analyzed and long-term follow-up was conducted. The follow-up period ended June 15, 2021, with the endpoint of all-cause death. Univariate Cox regression was used to analyze the influencing factors of all-cause death in patients, and Kaplan-Meier (K-M) survival curve was used to analyze the survival time of patients. At the same time, peripheral venous blood was collected from 27 DCM patients. After separating white blood cells, DNA was extracted, and whole exome sequencing was performed to screen potential pathogenic genes.Results:Among the 55 DCM patients, 40 were males and 15 were females. The age was (54.09 ± 12.38) years old. The heart function classification of New York Heart Association (NYHA) was mainly grade Ⅱ and Ⅲ, accounting for 94.55% (52/55). The follow-up time for 55 DCM patients was (7.02 ± 2.96) years, and 17 patients experienced all-cause death, accounting for 30.91% (17/55), including 15 males and 2 females. Compared with the survival group, the death group had a lower incidence of syncope (χ 2 = 6.57, P = 0.010), but higher rates of bilateral lower limb edema (χ 2 = 6.43, P = 0.017), pulmonary congestion (χ 2 = 7.61, P = 0.006), intraventricular conduction block (χ 2 = 6.41, P = 0.011), and angiotensin-converting enzyme inhibitor (ACEI) use (χ 2 = 6.57, P = 0.010), as well as increased left ventricular diameter ( t = 2.36, P = 0.022). Univariate Cox regression analysis showed that bilateral lower limb edema [hazard ratio ( HR) = 4.61, P = 0.042] and intraventricular conduction block ( HR = 3.20, P = 0.019) were risk factors for all-cause death of DCM patients. The results of K-M survival curve analysis showed that patients with bilateral lower limb edema and intraventricular conduction block had higher all-cause death rates (log-rank χ 2 = 5.02, 6.24, P = 0.025, 0.012). Whole exome sequencing results showed that 4 patients were detected to carry pathogenic or suspected pathogenic gene mutations, with a positive rate of 14.81% (4/27), involving three genes: β-myosin heavy chain 7 (MYH7), calreticulin 3 (CALR3), and gelsolin (GSN). Conclusions:The all-cause death rate of DCM patients in the Keshan disease area of Sichuan Province is relatively high. Dead patients are prone to bilateral lower limb edema, pulmonary congestion, and intraventricular conduction block, as well as increased left ventricular diameter. Bilateral lower limb edema and intraventricular conduction block are independent predictive risk factors for all-cause death in DCM patients. MYH7, CALR3 and GSN are involved in the pathogenesis of DCM.

Objective:To investigate the differences in polyunsaturated fatty acid metabolites in seminal plasma between normal group and low sperm motility group, and their relationship with sperm motility.Methods:Totally 581 human seminal plasmas collected from Guizhou Province were divided into normal group and low sperm motility group according to the motility rate. The metabolite products of 24 kinds of polyunsaturated fatty acids were detected by liquid phase tandem mass spectrometry/mass spectrometry (LC-MS/MS). Through the in vitro culture experiment of sperm, the culture supernatant was collected during the culture of the upstream screening sperm for 24 h, and the polyunsaturated fatty acid products with different differences detected in the different groups of seminal plasma were also verified by LC-MS/MS method. Results:Among the polyunsaturated fatty acid metabolites detected in seminal plasma, the concentrations of 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) in the seminal plasma of the low sperm motility group were significantly different from those in the normal seminal plasma group ( P=0.001, P=0.033). In vitro culture experiments showed that the concentration of 9-HODE and 13-HODE in the culture medium increased significantly with the prolongation of culture time and the decrease of sperm motility rate ( P=0.003, P=0.035). Statistical analysis showed that the concentrations of 9-HODE and 13-HODE were significantly negatively correlated with sperm motility （ r=-0.91， P=0.045； r=-0.95， P=0.026）. Considering the sperm density factor in the analysis of polyunsaturated fatty acid metabolites and sperm motility in the seminal plasma of the population, the results showed that 9-HODE and 13-HODE were significantly higher in the low sperm motility group than in the normal group ( P<0.000 1). And there was a negative correlation ( r=-0.38) with the sperm motility rate ( P<0.000 1). Conclusion:9-HODE and 13-HODE are released into the culture medium and seminal plasma by sperm cell metabolism, which is a potential indicator of the degree of sperm membrane damage, which may be helpful in judging and detecting the asthenospermia.

Objective:To investigate the differences in polyunsaturated fatty acid metabolites in seminal plasma between normal group and low sperm motility group, and their relationship with sperm motility.Methods:Totally 581 human seminal plasmas collected from Guizhou Province were divided into normal group and low sperm motility group according to the motility rate. The metabolite products of 24 kinds of polyunsaturated fatty acids were detected by liquid phase tandem mass spectrometry/mass spectrometry (LC-MS/MS). Through the in vitro culture experiment of sperm, the culture supernatant was collected during the culture of the upstream screening sperm for 24 h, and the polyunsaturated fatty acid products with different differences detected in the different groups of seminal plasma were also verified by LC-MS/MS method. Results:Among the polyunsaturated fatty acid metabolites detected in seminal plasma, the concentrations of 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) in the seminal plasma of the low sperm motility group were significantly different from those in the normal seminal plasma group ( P=0.001, P=0.033). In vitro culture experiments showed that the concentration of 9-HODE and 13-HODE in the culture medium increased significantly with the prolongation of culture time and the decrease of sperm motility rate ( P=0.003, P=0.035). Statistical analysis showed that the concentrations of 9-HODE and 13-HODE were significantly negatively correlated with sperm motility （ r=-0.91， P=0.045； r=-0.95， P=0.026）. Considering the sperm density factor in the analysis of polyunsaturated fatty acid metabolites and sperm motility in the seminal plasma of the population, the results showed that 9-HODE and 13-HODE were significantly higher in the low sperm motility group than in the normal group ( P<0.000 1). And there was a negative correlation ( r=-0.38) with the sperm motility rate ( P<0.000 1). Conclusion:9-HODE and 13-HODE are released into the culture medium and seminal plasma by sperm cell metabolism, which is a potential indicator of the degree of sperm membrane damage, which may be helpful in judging and detecting the asthenospermia.

Objective To establish several human umbilical vein endothelial cell ( HUVEC ) strains with over-ex-pression or low expression of receptor for activated C kinase 1 ( RACK1 ) , which will provide an effective tool for future studying the function of RACK1 in arrhythmia.Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP.At the same time, designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence , then subcloned into the plas-mid pGenesil-1 .The HUVEC cells were transfected with these plasmids and screened by using G 418 .And the expression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot , respectively . Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK 1 were constructed success-fully.After a 48 h transfection of HUVEC cells with the recombinant vectors and G 418 selection, the positive cell clones were obtained .qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhanced and knocked-down RACK1 expression in HUVEC strains .Conclusions HUVEC cell strains with over-expression and low expression of RACK 1 have been successfully established .