Based on the technology of platelet proteomics, the key regulatory proteins and pathogenesis of coronary heart disease with phlegm and blood stasis syndrome were explored and analyzed. Based on the previous laboratory research, the model of coronary heart disease in mini-swine with phlegm-stasis cementation syndrome was duplicated. The model was judged by the changes in blood lipid and myocardial tissue characteristics. Furthermore, the platelet proteins were studied by quantitative proteomics, and the differentially expressed proteins were screened. The critical regulatory proteins and biological pathways of coronary heart disease with phlegm-stasis cementation syndrome were analyzed by bioinformatics. After ten weeks of modeling, the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), very low density lipoprotein (VLDL-C), triglyceride (TG), creatine kinase (CK) and creatine kinase-MB (CK-MB) in the model group were significantly increased, reflecting the pathological changes such as increased blood lipid, abnormal coagulation function and myocardial ischemia in the model group. In addition, compared with the sham group, there were 26 up-regulated proteins and 8 down-regulated proteins in the platelets of the model group. Combined with bioinformatics analysis, it was found that differential proteins mainly involved in glycolysis/gluconeogenesis, pyruvate metabolism, lipid and atherosclerosis, Ras protein signal transduction. Among them, lactate dehydrogenase B (LDHB), alcohol dehydrogenase 5 (ADH5), neuroblastoma ratsarcoma viral oncogene homolog (NRAS) and Kirsten ratsarcoma viral oncogene homolog (KRAS) play a central role when interacting with other proteins and simultaneously participate in multiple action pathways. The results showed that LDHB, ADH5, NRAS, and KRAS may be the marker proteins in CHD with phlegm-stasis cementation syndrome by regulating glycolysis/gluconeogenesis, pyruvate metabolism, lipid and atherosclerosis, Ras protein signal transduction and other biological processes.

Anticoagulants ; Female ; Heparin ; Humans ; Pregnancy ; Pulmonary Embolism/diagnostic imaging* ; Thrombocytopenia

Aim To investigate the effects of FKBP38 gene on nonalcoholic fatty liver disease ( NAFLD ) model induced by methionine and choline deficiency j J diet (MCD) in mice.Methods The mutant model of hepatocellular specific deletion of FKBP38 gene was successfully established.The mice were divided into wild-type group ( WT) and homologous knockout group (L-FKBP38 ).Mice were fed with MCD for four weeks to construct NAFLD model.Liver injury was e- valuated by the contents of alanine transaminase (ALT), aspartate transaminase (AST) in the serum samples.We also performed HE staining, examined lipid accumulation by triglyceride (TG) and total cholesterol (CHO) and oil red staining, as well as macrophage infiltration by F4/80 immunohistochemical stai-ning of the liver sections.Fatty acid metabolism-relat ed genes were quantifier] by Quantitative Real-time PCR assays.Results Comparer] with WT group, the levels of ALT, AST, TG and CHO in serum signifi- eantly inereased ( P < 0.05 ) ; liver damage , lipid ac- eumulation, and maerophage infiltration were markedly more severe, and the expressions of fatty aeirl oxidation related genes PPARa, ACOX-1 , CPT-la and SIRT3 markedly rleereaserl ( P < 0.05) in the liver samples of L-FKBP38 group.Conclusions Hepatocellular speeifie deletion of FKBP38 intensifies lipid accumula- tion by inhibiting fatty aeid oxidation in the liver, thus exaeerbating nonaleoholie fatty liver disease.

ObjectiveThe absorption of substances into blood is mainly dependent on the mesenteric lymphatic pathway and the portal venous pathway. The substances transported via the portal venous pathway can be metabolized by the biotransformation in the liver. On the contrary, the substances in the mesenteric lymph fluid enter the blood circulation without biotransformation and can affect the body directly. Alcohol consumption is strongly linked to global health risk. Previous reports have analyzed the changes of metabolites in plasma, serum, urine, liver and feces after alcohol consumption. Whether alcohol consumption affects the metabolites in lymph fluid is still unknown. Therefore, it is particularly important to explore the changes of substances transported via the mesenteric lymphatic pathway and analyze their harmfulness after alcohol drinking. MethodsIn this study, male Wistar rats were divided into high, medium, and low-dosage alcohol groups (receiving Chinese Baijiu at 56%, 28% and 5.6% ABV, respectively) and water groups. The experiment was conducted by alcohol gavage lasting 10 d, 10 ml·kg^-1·d^-1. Then mesenteric lymph fluid was collected for non-targeted metabolomic analysis by using liquid chromatography-mass spectrometry (LC-MS) and bioinformatic analysis. Principal component analysis and hierarchical clustering were performed by using Biodeep. Meanwhile, KEGG enrichment analysis of the differential metabolites was also performed by Biodeep. MetaboAnalyst was used to analyze the relationship between the differential metabolites and diseases. ResultsThe metabolites in the mesenteric lymph fluid of the high-dosage alcohol group change the most. Based on the KEGG enrichment analysis, the pathways of differential metabolites between the high-dosage alcohol group and the control group are mainly enriched in the central carbon metabolism in cancer, bile secretion, linoleic acid metabolism, biosynthesis of unsaturated fatty acids, etc. Interestingly, in the biosynthesis of unsaturated fatty acids category, the content of arachidonic acid is increased by 7.25 times, whereas the contents of palmitic acid, oleic acid, stearic acid, arachidic acid and erucic acid all decrease, indicating lipid substances in lymph fluid are absorbed selectively after alcohol intake. It’s worth noting that arachidonic acid is closely related to inflammatory response. Furthermore, the differential metabolites are mainly related with schizophrenia, Alzheimer’s disease and lung cancer. The differential metabolites between the medium-dosage alcohol and the control group were mainly enriched in phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, linoleic acid metabolism and cholesterol metabolism. The differential metabolites are mainly related to schizophrenia, Alzheimer’s disease, lung cancer and Parkinson’s disease. As the dose of alcohol increases, the contents of some metabolites in lymph fluid increase, including cholesterol, L-leucine, fumaric acid and mannitol, and the number of metabolites related to schizophrenia also tends to increase, indicatingthat some metabolites absorbed by the intestine-lymphatic pathway are dose-dependent on alcohol intake. ConclusionAfter alcohol intake, the metabolites transported via the intestinal-lymphatic pathway are significantly changed, especially in the high-dosage group. Some metabolites absorbed via the intestinal-lymphatic pathway are dose-dependent on alcohol intake. Most importantly, alcohol intake may cause inflammatory response and the occurrence of neurological diseases, psychiatric diseases and cancer diseases. High-dosage drinking may aggravate or accelerate the occurrence of related diseases. These results provide new insights into the pathogenesis of alcohol-related diseases based on the intestinal-lymphatic pathway.

Aim To investigate the effects of CPD1,a novel phosphodiesterase 5 inhibitor,on renal pathological phenotype and fibrotic protein expression in renal fibrosis model mice. Methods Male C57BL/6 J mice were divided into three groups randomly(sham group,UUO group and UUO+CPD1 group). Unilateral ureteric obstruction model was constructed by surgery,and CPD1(5 mg·kg-1·d-1)was administered by intragastric administration two hours after the modeling for seven days. HE and Sirius Red staining were used to observe the distribution of tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of fibronectin(FN),α-SMA,collagen-I and kidney injury molecule-1(Kim-1). Results Compared with sham operation group,the renal tubules of mice were dilated and accompanied by a large amount of inflammatory infiltration. Moreover,the expressions of FN,α-SMA,collagen-I and Kim-1 proteins increased significantly(P<0.05)in UUO group. CPD1 treatment improved the kidney structure and decreased the expression of collagen fibers. Furthermore,CPD1 inhibited the expression of FN,α-SMA,collagen-I and Kim-1 markedly(P<0.05). Conclusions Phosphodiesterase 5 inhibitor CPD1 alleviates the progression of renal fibrosis induced by unilateral ureteral obstruction through down-regulating ECM deposition in the extracellular matrix and expression of Kim-1. The specific mechanism remains to be further studied.

Aim To investigate the effect of dietary intake of o)-3 poly unsaturated fatty acids ( u>-3 PUFAs) on the immune function of chronic graft versus host disease (cGVH) lupus model mice.Methods A single intraperitoneal injection of bml2 mice lymphocytes was used to establish a cGVH mouse model.On the day of modeling, 90% cd-3 PUFAs and 97% EPA were given by gavage for 14 days.The immune indexes of mice were evaluated by flow cytometry, and the serum total J J J ∗ IgG levels were measured by ELISA.Results Compared with control group, cGVH group significantly down-regulated Treg subsets, and up-regulated the Tfh , GC B and plasma subsets in the lupus mice.Comparer] with model control group, u>-3 PUFAs could significantly elevate Treg subsets, and decrease TFH, (X] B, and plasma subsets; serum total IgG levels in the 97% EPA group were significantly reduced.Conclusion In the cGVH lupus mouse model, co-3 PUFAs can suppress some immune functions by increasing Treg cells, reducing TFH, GC B, plasma cells and inhibiting the secretion of IgG.Such immunomodulatory effect provides new sights into the development of a potentially novel treatment modality for cGVH.

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*

This study was designed to evaluate the protective effect of CPD1, a novel phosphodiesterase 5 inhibitor, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury (UIRI). Male BALB/c mice were subjected to UIRI, and treated with CPD1 once daily (i.g, 5 mg/kg). Contralateral nephrectomy was performed on day 10 after UIRI, and the UIRI kidneys were harvested on day 11. Hematoxylin-eosin (HE), Masson trichrome and Sirius Red staining methods were used to observe the renal tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of proteins related to fibrosis. HE, Sirius Red and Masson trichrome staining showed that CPD1-treated UIRI mice had lower extent of tubular epithelial cell injury and deposition of extracellular matrix (ECM) in renal interstitium compared with those in the fibrotic mouse kidneys. The results from immunohistochemistry and Western blot assay indicated significantly decreased protein expressions of type I collagen, fibronectin, plasminogen activator inhibitor-1 (PAI-1) and α-smooth muscle actin (α-SMA) after CPD1 treatment. In addition, CPD1 dose-dependently inhibited the expression of ECM-related proteins induced by transforming growth factor β1 (TGF-β1) in normal rat kidney interstitial fibroblasts (NRK-49F) and human renal tubular epithelial cell line (HK-2). In summary, the novel PDE inhibitor, CPD1, displays strong protective effects against UIRI and fibrosis by suppressing TGF-β signaling pathway and regulating the balance between ECM synthesis and degradation through PAI-1.
Animals ; Humans ; Male ; Mice ; Rats ; Extracellular Matrix Proteins ; Fibrosis ; Kidney ; Kidney Diseases ; Phosphodiesterase 5 Inhibitors ; Plasminogen Activator Inhibitor 1

In order to investigate the effects of asiaticoside (Ass) on H9C2 cardiomyocytes, the present study examined the potential intervention of Ass on the proliferation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/Bcl-2 homology domain protein (Beclin-1) signaling pathway in H9C2 cardiomyocytes following oxygen and glucose deprivation/reperfusion (OGD/R) injury. H9C2 cardiomyocytes were selected as the research objects, and the activity of H9C2 was detected by cell counting kit-8 (CCK-8). H9C2 cells were divided into control group, OGD/R group, Ass low concentration group (10 μmol·L^-1), Ass high concentration group (80 μmol·L^-1) and Ass high concentration + chloroquine group (80 μmol·L^-1 + 50 μmol·L^-1). The control group was cultured under normal conditions, and the other groups were treated with oxygen and glucose deprivation for 4 h and reperfusion for 2 h. The activity and content of aspartic aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) in the supernatant of H9C2 cardiomyocytes were detected by enzyme-linked immunosorbent assay. Autophagy staining assay kit with monodansylcadaverine (MDC) method to observe cellular autophagy; molecular docking technique to identify the molecular targets of Ass. Immunofluorescence was used to observe the effect of the drug on cell number. The expression levels of PI3K, Akt, selective autophagy adaptor protein (P62) and Beclin-1 were detected by Western blot. Compared with OGD/R group, Ass group had a protective effect from 10-80 μmol·L^-1, and the activities and contents of AST, LDH and CK were decreased. The protein expression levels of PI3K, Akt, P62 and Beclin-1 were decreased. Compared with the administration group, the activities and contents of AST, LDH and CK in Ass high-concentration + chloroquine group were significantly decreased, and the protein expression levels of PI3K, Akt, Beclin-1 and P62 were significantly decreased. Immunofluorescence showed that the inhibitor group and each administration group had different degrees of protective effect compared with the model group. Asiaticoside can reduce the injury of H9C2 cardiomyocyte induced by OGD/R, reduce the content of AST, LDH and CK, reduce the expression level of P62 protein, and reduce autophagy, which may be closely related to the inhibition of PI3K/Akt/Beclin-1 signaling pathway activation.

The hyperacute stage of myocardial infarction refers to a period of time within 30 minutes after the occurrence of myocardial infarction, when the symptoms are not obvious and the diagnosis is difficult, and the related pathophysiological mechanism has received less attention. In this study, proteomics was used to investigate the pathological changes in the early hyperacute phase of myocardial infarction, aiming to provide experimental evidence for pathological mechanism of myocardial infarction hyperacute stage. Meanwhile, the intervention effect and related mechanism of salvianolate injection were discussed based on heat shock protein B6 (HSPB6), aiming to benefit the clinical rational use of salvianolate injection. The protein expression changes before and after myocardial infarction model establishment were detected by label-free proteomics via mass spectrometry and analyzed by bioinformatics method. Then the binding effect of salvianolate injection on the commonly differential protein HSPB6 was evaluated by molecular docking technology, which was finally verified by animal experiments. All animal experimental protocols were approved by the Ethics Committee of Xiyuan Hosptial (2022XLC041). The results of this study showed that a total of 2 166 proteins were quantified by lable-free proteomics, of which 194 shared differential proteins were involved in myocardial injury and body regulation in the hyperacute phase of myocardial infarction, mainly involving molecular functions such as protein homodimerization activity, oxygen binding and transport, and serine endopeptidase inhibitor activity. Among them, HSPB6 protein is involved in the regulation of myocardial function. Molecular docking results indicated that magnesium salvianolate acetate, which is the main component of salvianolate injection, had the lowest binding energy with HSPB6 protein: -14.53 kcal·mol^-1. Animal experiments showed that compared with the Sham group, the model group had significantly lower ejection fraction (EF) and fractional shortening (FS) (P < 0.001), cardiac blood perfusion decreased significantly (P < 0.001). There were obvious pathological changes such as myocardial fiber disorder, cardiomyocyte edema and interstitial small blood vessel congestion; the injury of cardiac function of rats in the administration group was attenuated, and the FS of rats in the low-dose group was significantly improved (P < 0.05), the pathological injury of myocardial tissue was markedly mitigated, and the expression of HSPB6 protein was up-regulated to varying degrees (P < 0.01, P < 0.001). In conclusion, salvianolate injection could be able to improve the cardiac function and pathological morphology of rats in the early hyperacute stage of myocardial infarction, and its mechanism may be related to the promotion of expression of HSPB6.