To investigate the effects and mechanisms of the water extract from Sorbus tianschanica(STE) on asthmatic airway inflammation, the mice were randomly divided into six groups, including a control group, a model group, a positive drug dexamethasone group(2 mg·kg~(-1)), a low-dose STE group(1 g·kg~(-1)), a medium-dose STE group(2 g·kg~(-1)), and a high-dose STE group(4 g·kg~(-1)). Except for the control group, all groups were subjected to ovalbumin induction to establish an asthma mouse model. The anti-inflammatory effects of STE were evaluated by examining pathological changes in lung tissue and measuring the levels of interleukin(IL)-4 and IL-5 in bronchoalveolar lavage fluid(BALF). Transcriptomic and proteomic methods were further employed to analyze differentially expressed genes and proteins, as well as their associated signaling pathways in lung tissue. Subsequently, the expression changes of key genes were verified by reverse transcription-quantitative polymerase chain reaction(RT-qPCR), and immunohistochemistry and Western blot methods were used to explore the regulatory mechanisms of STE in the pathogenesis of asthma in mice. Molecular docking was performed by using AutoDock Vina software to evaluate the binding affinity of the main active components in STE with the target proteins, including phosphatidylinositol-3-kinase catalytic subunit α(PIK3CA), Toll-like receptor 4(TLR4), protein kinase B1(Akt1), and matrix metallopeptidase 9(MMP9). The results showed significant inflammatory cell infiltration and fibrous tissue proliferation in the lung tissue of mice in the model group. However, these pathological changes were markedly reduced following STE intervention. Compared with those of the control group, the expression levels of IL-4 and IL-5 in the BALF of the model group were significantly increased but notably decreased following STE intervention. Transcriptomic and proteomic analyses identified key genes and proteins associated with allergic asthma, including tumor necrosis factor(TNF), IL-6, TLR4, PIK3CA, and MMP9. RT-qPCR validation revealed that high-dose STE intervention significantly downregulated the expressions of PIK3CA, IL-6, Akt1, MMP9, IL-13, nuclear factor-kappa B(NF-κB), TNF, CXC motif chemokine ligand 1(CXCL1), and TLR4 mRNAs and significantly upregulated the expression of signal transducer and activator of transcription 1(STAT1) mRNA. Western blot and immunohistochemical analyses confirmed that STE significantly downregulated the expressions of MMP9, TLR4, PIK3CA, and phosphorylated protein kinase B(p-Akt) in lung tissue of asthmatic mice. Moreover, molecular docking demonstrated that kaempferol-3,7-diglucoside, isoquercitrin, quercetin-3-gentiobioside, and hyperoside in STE exhibited stable binding affinities with PIK3CA, TLR4, Akt1, and MMP9, suggesting that the active components may exert anti-inflammatory effects by targeting and modulating asthma-related signaling pathways. In summary, STE exerts anti-asthmatic effects by inhibiting the expressions of PIK3CA, MMP9, p-Akt, and TLR4 and regulating the TLR4/PI3K/Akt/MMP9 signaling pathway.
Animals ; Asthma/metabolism* ; Toll-Like Receptor 4/metabolism* ; Signal Transduction/drug effects* ; Mice ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Mice, Inbred BALB C ; Drugs, Chinese Herbal/administration & dosage* ; Female ; Humans ; Lung/immunology* ; Male

Objective To investigate the effects of LBL-007, a novel fully human lymphocyte activation gene 3 (LAG-3) monoclonal antibody, in combination with programmed cell death protein 1 (PD-1) antibody, on the invasion, migration and proliferation of tumor cells, and to elucidate the underlying mechanisms. Methods Human lymphocyte cells Jurkat were co-cultured with A549 and MGC803 tumor cell lines and treated with the isotype control antibody human IgG, LBL-007, anti-PD-1 antibody BE0188, or tumor necrosis factor-alpha (TNF-α, the NF-κB signaling pathway agonist). Tumor cell proliferation was assessed using a colony formation assay; invasion was measured by Transwell^TM assay; migration was evaluated using a wound healing assay. Western blotting was employed to determine the expression levels of NF-κB pathway-related proteins: IκB inhibitor kinase alpha (Ikkα), phosphorylated Ikkα (p-IKKα), NF-κB subunit p65, phosphorylated p65 (p-p65), NF-κB Inhibitor Alpha (IκBα), phosphorylated IκBα (p-IκBα), matrix metalloproteinase 9 (MMP9), and MMP2. Results Compared with the control and IgG isotype groups, LBL-007 and BE0188 significantly reduced tumor cell proliferation, invasion, and migration. They also decreased the phosphorylation of p-IKKα, p-p65 and p-IκBα, and the expression of MMP9 and MMP2 of tumor cells in the co-culture system. The combined treatment of LBL-007 and BE0188 enhanced inhibitory effects. Treatment with the NF-κB signaling pathway agonist TNF-α reversed the suppressive effects of LBL-007 and BE0188 on tumor cell proliferation, invasion, migration, and NF-κB signaling. Conclusion LBL-007 and anti-PD-1 antibody synergistically inhibit the invasion, migration, and proliferation of A549 and MGC803 tumor cells by blocking the NF-κB signaling pathway.
Humans ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Neoplasm Invasiveness ; Antibodies, Monoclonal/pharmacology* ; Programmed Cell Death 1 Receptor/antagonists & inhibitors* ; Cell Line, Tumor ; Antigens, CD/immunology* ; Lymphocyte Activation Gene 3 Protein ; A549 Cells ; I-kappa B Kinase/metabolism* ; Jurkat Cells ; Matrix Metalloproteinase 9/metabolism*

OBJECTIVE: To explore the mechanism of action of asperosaponin VI (AVI) in the treatment of rheumatoid arthritis (RA) and validate it in ex vivo experiments using network pharmacology and molecular docking methods. METHODS: The predicted targets of AVI were obtained from PharmMaper, UniProt and SwissTarget Prediction platforms, the disease targets were collected from Online Mendelian Inheritance in Man, Therapeutic Target Database and GeneCards databases, the intersection targets of AVI and RA were obtained from Venny 2.1.0, and the protein-protein interaction (PPI) network was obtained from STRING database, which was analyzed by Cytoscape software and screened to obtain the core targets. Cytoscape software was used to analyze PPI network and screen the core targets. Based on the Database for Annotation, Visualization and Integrated Discovery database, Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed, and Cytoscape software was used to construct the "Disease-Pathway-Target-Drug" network, which was finally verified by molecular docking and animal experiments. RESULTS: Network pharmacological studies showed that AVI was able to modulate 289 targets, with 102 targets for the potential treatment of RA, with the core pathway being the AKT/PI3K signaling pathway, and the core targets being the epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9). Molecular docking results showed that AVI could produce strong binding with both of the 2 core targets. In vitro cellular experiments showed that AVI reduced nitric oxide, prostaglandin E₂, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1 β levels (P<0.05) and inhibited cyclooxygenase-2, nitric oxide synthase, EGFR, MMP9, phosphorylated phosphoinositide 3-kinase (p-PI3K), and phosphorylated serine-threonine kinase (p-AKT) proteins (P<0.05). The results of in vivo studies showed that AVI improved RA score and foot swelling thickness and decreased TNF-α, IL-6, p-PI3K and p-AKT levels in RA rats (P<0.05). CONCLUSION AVI exerts anti-inflammatory and anti-RA effects which might be related to the EGFR/MMP9/AKT/PI3K pathway.
Saponins/chemistry* ; ErbB Receptors/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Animals ; Arthritis, Rheumatoid/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Network Pharmacology ; Male ; Rats

OBJECTIVE: To investigate the effect of midazolam on pain in lumbar disc herniation model rats based on p38 MAPK signaling pathway. METHODS: Fifty SPF-grade Sprague-Dawley healthy rats, half male and half female, were selected and randomly divided into normal group, model group, and low-dose, medium-dose, high-dose groups. Model group and low-dose, medium-dose, high-dose groups were initially modeled for lumbar disc herniation. Intraperitoneal injection of saline was performed in rats of normal and model groups; and in the low-dose, medium-dose, and high-dose groups, intraperitoneal injection of midazolam was performed with doses of 30, 60, and 90 mg/kg, respectively. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), substance P (SP), neuropeptide Y (NPY) were detected in the serum of rats by enzyme-linked immunoassay. The expression of p38 MAPK and matrix metalloproteinase-3(MMP-3) protein were detected by Western blot in the tissues of rats of each group. RESULTS: The levels of TNF-α, IL-1β and β-EP were higher and the level of 5-HT was lower in the model group than in the normal group(P<0.05);the levels of TNF-α, IL-1β and β-EP were lower and the level of 5-HT was higher in the low-dose, medium-dose and high-dose groups than in the model group(P<0.05). The levels of SP and NPY increased in the model group compared with the normal group (P<0.05) and the levels of SP and NPY decreased in the low-dose, medium-dose and high-dose groups compared with the model group (P<0.05). The expression of p38 MAPK and MMP-3 increased in the model group compared with the normal group (P<0.05); the expression of p38 MAPK and MMP-3 decreased in the low-dose, medium-dose and high-dose compared with the model group(P<0.05). CONCLUSION Midazolam may ameliorate the immune inflammatory response in rats with a model of lumbar disc herniation, possibly regulated through the p38MAPK signaling pathway.
Rats ; Male ; Female ; Animals ; Intervertebral Disc Displacement/pathology* ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 3/metabolism* ; Midazolam ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; MAP Kinase Signaling System/physiology* ; Pain ; p38 Mitogen-Activated Protein Kinases/metabolism*

OBJECTIVE: To investigate the effect of electroacupuncture on osteoarthritis in rats and explore the possible mechanism. METHODS: Thirty SD rats were randomly divided into osteoarthritis model group, electro-acupuncture group and control group (n=10), and in the former two groups, early osteoarthritis was induced using a modified DMM surgical modeling method. After successful modeling, the rats in the electro-acupuncture group were treated with electro-acupuncture at bilateral "Housanli" and "Anterior knee point". Behavioral tests of the rats were performed and scored using the LequesneMG scale. Subchondral bone degeneration was observed in each group, and serum levels of IL-1β, ADAMTS-7, MMP-3 and COMP were measured using ELISA. The mRNA and protein expressions of IL-1β, Wnt-7B, β-catenin, ADAMTS-7, and MMP-3 in the cartilage tissue of the knee joints were detected using RT-PCR and Western blotting. RESULTS: In behavioral tests, the rats in the model and electroacupuncture groups had significantly higher LequesneMG scores after modeling than those in the control group (P < 0.05). After 20 days of treatment, LequesneMG scores were significantly lowered in rats in the electroacupuncture as compared with the model rats (P < 0.05). Imaging examination revealed obvious subchondral bone damage in both the electroacupuncture group and the model group, but the damages were significantly milder with former group. Compared with the model rats, the rats receiving electroacupuncture had significantly lower serum levels of IL-1β, ADAMTS-7, MMP-3 and COMP (P < 0.05) with also lower expressions of IL-1β, Wnt-7B, β-catenin, ADAMTS-7 and MMP-3 in the cartilage tissues at both the mRNA and protein levels (P < 0.05). CONCLUSION Electroacupuncture can alleviate joint pain and improve subchondral bone damage in rats with osteoarthritis by reducing IL-1β levels in the joint cartilage tissue and serum to alleviate joint inflammation and by reducing such cytokines as ADAMTS-7 and MMP-3 via regulating the Wnt-7B/β-catenin signaling pathway.
Rats ; Animals ; Electroacupuncture ; Matrix Metalloproteinase 3/metabolism* ; Rats, Sprague-Dawley ; beta Catenin/metabolism* ; Osteoarthritis/metabolism* ; Wnt Signaling Pathway ; Cartilage, Articular ; Inflammation/metabolism*

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.
Animals ; Mice ; Apolipoproteins E ; Atherosclerosis/genetics* ; Beclin-1 ; Cholesterol, LDL ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Plaque, Atherosclerotic ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/genetics* ; Vascular Cell Adhesion Molecule-1/genetics*

OBJECTIVES: The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy. METHODS: The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO₂ dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO₂ group, a LY294002 group, a SC79 group, a LY294002+SiO₂ group, and a SC79+SiO₂ group were set up in this experiment. Macrophages in the LY294002+SiO₂ group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO₂ group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO₂ (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting. RESULTS: After the macrophages were exposed to SiO₂ dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO₂ concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO₂ dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO₂ (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO₂ group (all P<0.05). Compared with the SiO₂ group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO₂ group. Compared with the SiO₂ group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO₂ group. CONCLUSIONS Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Silicon Dioxide/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Matrix Metalloproteinase 1/metabolism* ; Tissue Inhibitor of Metalloproteinase-1 ; Sirolimus ; Beclin-1/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Dust ; TOR Serine-Threonine Kinases/metabolism* ; Lung/metabolism* ; Fibroblasts/metabolism* ; Silicosis/metabolism* ; Macrophages/metabolism* ; Autophagy

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.
Humans ; Matrix Metalloproteinase 2/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; beta Catenin/metabolism* ; Vimentin/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Cell Line, Tumor ; Wnt Signaling Pathway ; Cadherins/genetics* ; Melanoma/genetics* ; Cyclin D/metabolism* ; Cell Proliferation ; Boraginaceae/genetics* ; RNA, Messenger ; Cell Movement

Jiming Powder is a traditional ancient prescription with good therapeutic effect in the treatment of heart failure, but its mechanism lacks further exploration. In this study, a mouse model of coronary artery ligation was used to evaluate the effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction. The study constructed a mouse model of heart failure after myocardial infarction using the method of left anterior descending coronary artery ligation. The efficacy of Jiming Powder was evaluated from multiple angles, including ultrasound imaging, hematoxylin-eosin(HE) staining, Masson staining, Sirius Red staining, and serum myocardial enzyme spectrum detection. Western blot analysis was performed to detect key proteins involved in ventricular remodeling, including transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), wingless-type MMTV integration site family member 3a(Wnt3a), β-catenin, matrix metallopeptidase 2(MMP2), matrix metallopeptidase 3(MMP3), TIMP metallopeptidase inhibitor 1(TIMP1), and TIMP metallopeptidase inhibitor 2(TIMP2). The results showed that compared with the model group, the high and low-dose Jiming Powder significantly reduced the left ventricular internal diameter in systole(LVID;s) and diastole(LVID;d), increased the left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), effectively improved cardiac function in mice after myocardial infarction, and effectively reduced the levels of myocardial injury markers such as creatine kinase(CK), creatine kinase isoenzyme(CK-MB), and lactic dehydrogenase(LDH), thus protecting ischemic myocardium. HE staining showed that Jiming Powder could attenuate myocardial inflammatory cell infiltration after myocardial infarction. Masson and Sirius Red staining demonstrated that Jiming Powder effectively inhibited myocardial fibrosis, reduced the collagen Ⅰ/Ⅲ ratio in myocardial tissues, and improved collagen remodeling after myocardial infarction. Western blot results showed that Jiming Powder reduced the expression of TGF-β1, α-SMA, Wnt3a, and β-catenin, decreased the levels of MMP2, MMP3, and TIMP2, and increased the level of TIMP1, suggesting its role in inhibiting cardiac fibroblast transformation, reducing extracellular matrix metabolism in myocardial cells, and lowering collagen Ⅰ and α-SMA content, thus exerting an anti-myocardial fibrosis effect after myocardial infarction. This study revealed the role of Jiming Powder in improving ventricular remodeling and treating myocardial infarction, laying the foundation for further research on the pharmacological effect of Jiming Powder.
Mice ; Animals ; Transforming Growth Factor beta1/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; beta Catenin/metabolism* ; Matrix Metalloproteinase 3/therapeutic use* ; Powders ; Ventricular Remodeling ; Stroke Volume ; Ventricular Function, Left ; Myocardial Infarction/drug therapy* ; Myocardium/pathology* ; Heart Failure/metabolism* ; Collagen/metabolism* ; Creatine Kinase ; Fibrosis

OBJECTIVE: To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA. METHODS: Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2, 5, and 10 ?M) for 24, 48, and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1β (IL-1β)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5, MMP3 and Col2a1. Further, the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot, and then, the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs. RESULTS: The 10 μM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells, and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1β, and reduced the effect of IL-1β on ADAMTS-5, and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs, including miR-940 and miR-576-5p. Then, the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group, the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs. CONCLUSION Salvianolic acid A attenuated the IL-1β-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.
Humans ; Apoptosis ; Cartilage/metabolism* ; Chondrocytes/metabolism* ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 3/metabolism* ; MicroRNAs/metabolism*