[Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway].

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

OBJECTIVE: To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor ß1/Smad (TGF-ß1/Smad) signaling pathway. METHODS: Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-ß1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-ß1/Smad signaling pathway. RESULTS: A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-ß signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss.

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

Identification of Hedyotis diffusa Willd-specific mRNA-miRNA-lncRNA network in rheumatoid arthritis based on network pharmacology, bioinformatics analysis, and experimental verification.

Hedyotis diffusa Willd (HDW) possesses heat-clearing, detoxification, anti-cancer, and anti-inflammatory properties. However, its effects on rheumatoid arthritis (RA) remain under-researched. In this study, we identified potential targets of HDW and collected differentially expressed genes of RA from the GEO dataset GSE77298, leading to the construction of a drug-component-target-disease regulatory network. The intersecting genes underwent GO and KEGG analysis. A PPI protein interaction network was established in the STRING database. Through LASSO, RF, and SVM-RFE algorithms, we identified the core gene MMP9. Subsequent analyses, including ROC, GSEA enrichment, and immune cell infiltration, correlated core genes with RA. mRNA-miRNA-lncRNA regulatory networks were predicted using databases like TargetScan, miRTarBase, miRWalk, starBase, lncBase, and the GEO dataset GSE122616. Experimental verification in RA-FLS cells confirmed HDW's regulatory impact on core genes and their ceRNA expression. We obtained 11 main active ingredients of HDW and 180 corresponding targets, 2150 RA-related genes, and 36 drug-disease intersection targets. The PPI network diagram and three machine learning methods screened to obtain MMP9, and further analysis showed that MMP9 had high diagnostic significance and was significantly correlated with the main infiltrated immune cells, and the molecular docking verification also showed that MMP9 and the main active components of HDW were well combined. Next, we predicted 6 miRNAs and 314 lncRNAs acting on MMP9, and two ceRNA regulatory axes were obtained according to the screening. Cellular assays indicated HDW inhibits RA-FLS cell proliferation and MMP9 protein expression dose-dependently, suggesting HDW might influence RA's progression by regulating the MMP9/miR-204-5p/MIAT axis. This innovative analytical thinking provides guidance and reference for the future research on the ceRNA mechanism of traditional Chinese medicine in the treatment of RA.

Modulation of mammary tumour progression using murine model by ethanol root extract of Saussurea costus (falc.) lipsch.

ETHNOPHARMACOLOGICAL RELEVANCE: Breast cancer is a major cause of death among human females across the globe. The anti-neoplastic agents or therapies used for the treatment of cancers can enhance longevity but are subsequently observed to deteriorate the quality of life due to the extensive side effects produced. Saussurea costus is a potential medicinal plant of the Himalayas with noticeable ethnopharmacological properties. The phytochemicals present in Saussurea costus are responsible for anti-carcinogenic potential and warranted nil or minimal side effects of Saussurea costus and directed to use this plant as a preventive or therapeutic drug candidate against cancers. AIM OF THE STUDY: The present study was planned to evaluate the anti-neoplastic activity of Saussurea costus root extract (SL) in rat mammary tumour model. MATERIALS AND METHODS: The anti-neoplastic activity of SL root extract at 3 different doses (100, 250 and 500 mg/kg BW) for 18 weeks against 12-dimethylbenz (a) anthracene (DMBA)-induced mammary tumours in Sprague Dawley (SD) female rats was analyzed through serum biochemistry (ALT, AST, ALP, Total protein, Creatinine and BUN), oxidative stress parameters (Lipid peroxidation, Catalase and Reduced glutathione), pro-inflammatory cytokines (TNF-α and NF-κB), immunohistochemical markers (Ki-67, MMP-9 and VEGF), real-time PCR (PCNA, p53, bax, bcl-2 and caspase-3, genes) and molecular docking. RESULTS: Inhibition of tumour parameters, minimal alteration in the liver (ALT, AST and ALP) and kidney enzymes (Creatinine and BUN), decreased activity of MDA, elevated levels of GSH and catalase, reduction in the levels of pro-inflammatory cytokines i.e. TNF-α and NF-κB, reduced gross and histomorphological changes, declined expression of Ki-67, MMP-9 and VEGF in vivo rat model, mRNA expression of cancer-related genes and docking of dehydrocostus lactone and costunolide with NF-κB and TNF-α demonstrated the chemopreventive action of SL root extract. CONCLUSIONS: The in-vivo trial elucidates anti-neoplastic activity of Saussurea costus root extract as demonstrated through the reduction of biochemical indices, oxidative stress parameters, histological changes, pro-inflammatory cytokines (NF-κB and TNF-α), cellular proliferation (Ki-67), metastases (MMP-9) and neovascularization (VEGF) markers with highest anti-neoplastic effect of SL extract at the dose of 500 mg/kg body weight. Therefore, the present study signifies the need to use the active principles present in the root extract of Saussurea costus against breast cancer as a therapeutic regimen.

Spent hops extract (Humulus Lupulus L.) attenuates inflammation and angiogenesis of the retina via the nuclear factor-kappaB and protein kinase B/extracellular signal-regulated kinase pathways.

Spent hops extract (SHE) is a plant extract containing compounds with proven anti-inflammatory and anti-angiogenic activities. However, extract may exert synergic effects compared to its individual polyphenol components. Inflammatory diseases of the retina may lead to visual impairment, a reduction of the comfort of life, and even blindness due to the formation of new pathological blood vessels. More effective therapeutic options are being sought. The goal of the present study was to investigate the anti-inflammatory and anti-angiogenic potentials of SHE on human retinal pigment epithelial cells (ARPE-19) stimulated by lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The SHE (250 µg/mL) was found to downregulate the gene expression of interleukin 6 (IL-6) to 33% in LPS-triggered cells; it also reduced both matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) mRNA expression to 13% and 43% respectively, and their activity to 82% (MMP-2) and 57% (MMP-9), compared to TNF-α-stimulated cells. Also, SHE modulated the TNF-α-induced expression of vascular endothelial growth factor (VEGF) and endothelial growth factor receptor 2 (VEGFR2). It is possible that SHE inhibited retinal inflammation and angiogenesis by suppressing the nuclear factor kappa B (NF-κB), protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways. Our results demonstrate that SHE has anti-inflammatory and anti-angiogenic potential against retinal diseases. This is the first such study to report on the efficacy of SHE on retinal inflammatory diseases.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

Combined dandelion extract and all-trans retinoic acid induces cytotoxicity in human breast cancer cells.

Breast cancer is one of the most prevalent and deadly cancers among women worldwide. Recently, natural compounds have been widely used for the treatment of breast cancer. Present study evaluated antiproliferative and anti-metastasis activities of two natural compounds of dandelion and all-trans-retinoic acid (ATRA) in human MCF-7 and MDA-MB231 breast cancer cells. We also evaluated the expression of MMP-2, MMP-9, IL-1ß, p53, NM23 and KAI1 genes. Data showed a clear additive cytotoxic effect in concentrations of 40 µM ATRA with 1.5 and 4 mg/ml of dandelion extract in MCF-7 and MDA-MB231 cells, respectively. In both cell lines, compared with the untreated cells, the expression levels of MMP-9 and IL-1ß were significantly decreased while p53 and KAI1 expression levels were increased. Besides, MMP-2 and NM23 had different expressions in the two studied cell lines. In conclusion, dandelion/ATRA co-treatment, in addition to having strong cytotoxic effects, has putative effects on the expression of anti-metastatic genes in both breast cancer cells.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200 µg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Salvianolic acid B promotes the invasion and migration of HO-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway.

OBJECTIVE: To elucidate the regulatory effects of salvianolic acid B (SalB) on trophoblast cells in preeclampsia (PE). METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenylte-trazolium bromide (MTT) assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by HO following treatment with different concentrations of SalB. The levels of oxidative stress-related molecules, including superoxide dismutase, glutathione-Px and malondialdehyde were detected using corresponding kits. Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, and the expression of apoptosis-related proteins was detected using western blot analysis. In the present study, wound healing and Transwell assays were performed to measure the levels of cell invasion and migration. Western blot analysis was also used to detect the expression levels of epithelial-mesenchymal transition-related proteins. The mechanisms underlying SalB were further investigated using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis, to determine the expression levels of matrix metallopeptidase 9 (MMP-9) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt). RESULTS: SalB increased the activity of HTR-8/Svneo cells, inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by HO. Furthermore, the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased. The pathway agonist, LY294002, and MMP-9 inhibitor, GM6001, reversed the effects of SalB on HO-induced cells. CONCLUSIONS: SalB promoted the invasion and migration of HO-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 the PI3K/Akt signaling pathway.

[Regulatory effect of moxibustion on LTB4/MMP-9 in serum of patients with rheumatoid arthritis].

OBJECTIVE: To observe the effects of moxibustion on the contents of leukotriene B4 (LTB4), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase -9 (MMP-9) in serum, and explore the protection mechanisms of moxibustion in the patients with rheumatoid arthritis (RA). METHODS: A total of 64 patients with RA were randomly divided into treatment group (n=31) and control group (n=33). The patients in the control group were treated with conventional medication for consecutive 5 weeks. Based on the treatment in the control group, the patients in the treatment group were treated with moxibustion at bilateral Shenshu (BL23), Zusanli (ST36) and Ashi points, 3 times a week, for consecutive 5 weeks. Separately, the visual analogue scale (VAS) score, morning stiffness score, the number of tender joints, the number of swollen joints, the score of the disease activity score of 28 joints (DAS28) were observed; the contents of rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and C-reative protein (CRP) in serum were determined by biochemical method; and the contents of LTB4, IL-17, TNF-α and MMP-9 in serum were detected by using ELISA before and after treatment in the patients of both groups. RESULTS: After treatment, VAS score, morning stiffness score, the number of tender joints, the number of swollen joints, DAS28 score, the contents of serum RF in both groups, and contents of serum CRP, ESR, LTB4, IL-17, TNF-α and MMP-9 in the treatment group were significantly reduced when compared with those before treatment (P<0.01, P<0.05). After treatment, VAS score, morning stiffness score, the number of tender joints, the number of swollen joints, DAS28 score, and the levels of LTB4, IL-17 and MMP-9 in serum were obviously lower in the treatment group when compared with the control group (P<0.01, P<0.05). In the treatment group, the changes before and after treatment in the levels of LTB4, IL-17 and TNF-α were positively correlated with that of MMP-9 (P<0.05, r>0). CONCLUSION: Moxibustion at BL23 and ST36 combined with conventional medication significantly relieves joint pain and reduce disease activity in RA patients, which may be related to the modulation of LTB4, IL-17 and MMP-9 by moxibustion.

1-Tetracosanol isolated from the leaves of Eupatorium glandulosum, accelerates wound healing by expressing inflammatory cytokines and matrix metalloproteinase.

ETHNOPHARMACOLOGICAL RELEVANCE: The leave paste of the plant, Eupatorium glandulosum H. B & K, has been traditionally used to treat cuts and wounds by the tribal community of the Nilgiris district of Tamilnadu, India. AIM OF THE STUDY: The present study was carried out to investigate the wound healing potential of this plant extract and the compound, 1-Tetracosanol, isolated from the ethyl acetate fraction. MATERIALS AND METHODS: An in vitro study was designed to compare the viability, migration and apoptosis of the fresh methanolic extract fractions and 1-Tetracosanol using mouse fibroblast NIH3T3 cell lines and human keratinocytes HaCaT cell lines, respectively. 1-Tetracosanol was evaluated for its viability, migration, qPCR analysis, in silico, in vitro and in vivo. RESULTS: 1-Tetracosanol at the concentration of 800, 1600, 3200 µM has significant wound closure of 99% at 24 h. The compound when screened in silico against various wound healing markers, TNF-α, IL-12, IL-18, GM-CSF and MMP-9, revealed high binding energy of -5, 4.9 and -6.4 kcal/mol for TNF-α, IL-18 and MMP-9, respectively. Gene expression and the release of cytokines increased at an early stage of the wound repair. 1-Tetracosanol, at 2% gel showed 97.35 ± 2.06% wound closure at 21st day. CONCLUSION: 1-Tetracosanol is a good lead for drug development targeted towards wound healing activity and work in this direction is in progress.

Effects and mechanism of Compound Qidan Formula on rats with HFpEF induced by hypertension and diabetes mellitus based on Ang â ¡/TGF-ß1/Smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Qidan Formula is composed of traditional Chinese herbs and has a good curative effect in the clinical application of cardiovascular diseases such as heart failure. However, its potential molecular mechanisms of action remain highly unknown. AIM OF THE STUDY: To observe the effect of Compound Qidan Formula on cardiac function in rats with HFpEF induced by hypertension and diabetes mellitus, and to explore its mechanism from Ang Ⅱ/TGF-ß1/Smads signaling pathway. MATERIALS AND METHODS: A total of 50 SPF-grade spontaneously hypertensive rats (SHR) aged 14 weeks, fed with a high-fat and high-sucrose diet for 16 weeks, and after 2 weeks of a high-fat and high-sucrose diet, 1% streptozotocin (25 mg/kg body weight)was injected intraperitoneally to establish a rat model of HFpEF induced by hypertension and diabetes mellitus. After 8 weeks of intragastric administration, the changes in cardiac morphology and function were evaluated by echocardiography after anesthesia; the heart tissue was taken and embedded in paraffin for Masson staining, and the pathomorphological changes of left atrial tissue were observed under the optical microscope; the mRNA transcription levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, MMP-9 and TIMP-1in left atrial tissue of rats were detected by RT-PCR; and the protein expressions were detected by Western blot. RESULTS: Compared with the SHR-DM group, the QD-Low and QD-High groups significantly decreased the left atrial (LA) anteroposterior diameter and interventricular septal thickness (IVST) and improved the peak velocity of mitral valve blood flow in early diastolic period (E), maximum mitral valve blood flow in systolic period (A), mitral ring myocardial movement velocity in early diastolic period (e') and E/e' ratio; the QD-High group significantly improved the E/A ratio, left atrial ejection fraction (LAEF) and left ventricular ejection fraction(LVEF). Masson staining showed that compared with the WKY group, the SHR-DM group had obvious myocardial histomorphological lesions. Compared with the SHR-DM group, the Compound Qidan Formula groups significantly improved cardiomyocyte hypertrophy and disordered arrangement and inhibited myocardial fibrosis; the mRNA expression levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, and MMP-9 in myocardial tissue of Compound Qidan Formula groups were significantly decreased, and the mRNA expression level of TIMP-1 was significantly increased. The protein expression levels of Ang Ⅱ, TGF-ß1, P-Smad2/3, and MMP-9 were significantly decreased. CONCLUSION: Compound Qidan Formula, composed of traditional Chinese herbs, can significantly improve cardiac function, improve atrial and ventricular remodeling, and prevent myocardial fibrosis and hypertrophy in rats with HFpEF induced by hypertension and diabetes mellitus. The mechanism may be related to regulating the Ang Ⅱ/TGF-ß1/Smad2/3 signaling pathway.

Activation of estrogen receptor α inhibits TLR4 signaling in macrophages and alleviates the instability of atherosclerotic plaques in the postmenopausal stage.

Acute cardiovascular events increase significantly in postmenopausal women. The relationship between estrogen receptor (ER) and plaque stability in the postmenopausal stage remains to be elucidated. We aimed to explore whether ERα activation improves plaque instability in the postmenopausal stage. Here, we report that postmenopausal women showed increased macrophage activation and plaque instability with increased MCP-1, MMP9, TLR4, MYD88 and NF-κB p65 and decreased ERα and TIMP1 expression in the vascular endothelium. Moreover, ovariectomy in LDLR-/- mice resulted in a significant increase in plaque area and necrotic core area, as well as a significant decrease in collagen content and an increase in macrophage accumulation in the artery. Ovariectomy also reduced serum estrogen levels and ERα expression and upregulated TLR4 and MMP9 expression in arteries in LDLR-/- mice. Estrogen or phytoestrogen therapy upregulated the expression level of ERα in ovariectomized mice and increased plaque stability by inhibiting macrophage accumulation and TLR4 signaling. In vitro, LPS incubation of RAW264.7 cells resulted in a significant decrease in ERα and TIMP1 expression and an increase in TLR4 activation, and estrogen or phytoestrogen treatment increased ERα and TIMP1 expression and inhibited TLR4 activation and MMP9 expression in LPS-treated RAW264.7 cells. Compared to control siRNA transfected RAW264.7 cells, TLR4 siRNA promoted TIMP1 expression in RAW264.7 cells with LPS incubation, but did not affect ERα expression in RAW264.7 cells with or without LPS treatment. The ERα inhibitor MPP abolished the regulatory effect of estrogen or phytoestrogen on LPS-induced RAW264.7 cells. In conclusion, the present study demonstrates that decreased ERα expression promotes macrophage infiltration and plaque instability in the postmenopausal stage, and activation of ERα in the postmenopausal stage alleviates atherosclerotic plaque instability by inhibiting TLR4 signaling and macrophage-related inflammation.

[Mechanism of total flavonoids of Ziziphora clinopodioides in improving atherosclerosis by regulating PI3K/Akt/mTOR pathway].

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.

Pilose antler (Cervus elaphus Linnaeus) polysaccharide and polypeptide extract inhibits bone resorption in high turnover type osteoporosis by stimulating the MAKP and MMP-9 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Pilose antler is a traditional Chinese medicine used to improve kidney function, strengthen tendons and bones, and prolong life, among other uses. It is widely employed in the treatment of osteoporosis. However, the molecular mechanisms underlying the treatment of high turnover osteoporosis are not fully understood. AIM OF THE STUDY: The present study aimed to investigate the molecular mechanism underlying pilose antler polysaccharide and polypeptide extracts in inhibiting bone resorption in high turnover osteoporosis, and compare the effects of the two components alone and in combination to explore whether they could produce synergistic enhancement effects. MATERIALS AND METHODS: The quantitative and qualitative characteristics of pilose antler polysaccharide and polypeptide extracts were detected by UV-visible spectrophotometry and high-performance liquid chromatography. A rat model of retinoic acid-induced osteoporosis was used to evaluate the inhibitory effect of the extracts on bone resorption. Enzyme-linked immunosorbent assay (ELISA) was used to detect the activity of factors related to high turnover type osteoporosis in rat serum. Western blotting was used to detect the expression of proteins related to the MAKP and MMP-9 signaling pathways in rat femurs. Fluorescence quantitative PCR was used to detect the transcription levels of genes related to the MAKP and MMP-9 signaling pathways in rat femur tissues. Hematoxylin and eosin staining were used to observe the osteoprotective effects of pilose antler polysaccharides and polypeptides. RESULTS: The yield of pilose antler polysaccharides was 8.3%, and was mainly composed of mannose, glucosamine hydrochloride, glucuronic acid, Galacturonic acid, Galactose hydrochloride, glucose, and galactose. The yield of the polypeptides was 26.2%, and eighty percent of the molecular weight of the antler polypeptides was 1.6 kDa-7kD, among which, the molecular weight of 7kD peptide accounted for 52% of the total. Both polysaccharides and peptides could reduce the activities of TRACP, OCN, ERK1, JNK, and MMP-9 in rat serum and reduce both the protein expression and gene transcription levels of ERK1, JNK, and MMP-9 in rat femur tissue with significant differences compared with the model group. Both extracts exerted significant protective effects on rat femur tissue. The effect of pilose antler polypeptides alone was better than that of polysaccharides either alone or in combination. CONCLUSIONS: Pilose antler polysaccharides, polypeptides, and their mixtures could inhibit the occurrence of bone resorption of high turnover osteoporosis by stimulating the MAKP and MMP-9 signaling pathways to reduce the expression of the ERK1, JNK, and MMP-9 genes and proteins, and could help alleviate bone loss caused by retinoic acid. Pilose antler polypeptides had a stronger effect on inhibiting bone resorption. The combination of the two components did not show synergistic enhancement effect, and the polysaccharide tended to moderate the inhibitory enhancement effect of the polypeptide.

Mechanism of astragalus injection to relieve symptoms of preeclampsia rat model by inhibiting MMP-9/sFlt-1/TNF-α.

Objective: The aim of this study was to observe the effect of astragalus injection on rats with preeclampsia. Methods: A total of 30 pregnant Sprague Dawley (SD) rats were randomly assigned to the model group (MG), the astragalus group (AG) or the control group (CG), with 10 rats in each group. The rat model of preeclampsia was established by subcutaneous injection of 50 mg/(kg∙d) of N-nitro-L-arginine methyl ester (L-NAME), and 0.024 ml/(g∙d) astragalus injection was administered intraperitoneally. The arterial pressure, urinary protein, placental mass, fetal weight, inflammatory factors in peripheral blood of pregnant rats, protein and mRNA levels of nuclear factor- κB (NF-κB), matrix metalloproteinase-9 (MMP-9), nuclear transcription factor 5 (NFAT-5), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and reactive oxygen species (ROS) activity, malondialdehyde (MDA) and nitric oxide (NO) levels in placental tissues were compared in the 3 groups. Results: After treatment, the arterial pressure and urinary protein levels in pregnant rats in the MG group were significantly higher than in the CG and AG groups (P < .05). The placental mass in the MG group was lower than in the CG and AG groups (P < .05). The messenger RNA (mRNA) and protein levels of sFlt-1, NFAT-5 and NF-κB, as well as ROS activity, MDA, inerleukin (IL)-6, tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) in the AG group were significantly lower than in the MG group, and mRNA and protein expression of MMP-9 and PlGF, as well as the NO level in the AG group, were significantly higher than in the MG (P < .05). Conclusions: Astragalus injection can effectively inhibit the expression of sFlt-1, NFAT-5, NF-κB and enhance the expression of PlGF and MMP-9 in the placental tissue of rats with preeclampsia, which may be the mechanism of preeclampsia treatment.

[Warm acupuncture stimulation improves cartilage damage and motor function by regulating JAK2/STAT3 signaling pathway in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of warm acupuncture on the expression of Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and inflammatory factors of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its underlying mechanisms in improving KOA. METHODS: New Zea-land rabbits were randomly divided into blank, model, warm acupuncture and medication groups (12 rabbits in each group). The KOA model was prepared by using the right hind limb tubular plaster extension fixation method. The rabbits in the warm acupuncture group received acupuncture of "Neixiyan"(EX-LE4),"Waixiyan"(ST35),"Heding"(EX-LE2) and "Zusanli"(ST36), followed by attaching an ignited moxa-stick segment to the acupuncture-handle. The treatment was conducted for 15 min, once a week for 4 weeks. The rabbits in the medication group received gavage of diclofenac sodium solution(0.35 mg/kg), once daily for 4 weeks. The dysfunction severity state of the rabbit's knee-joint was evaluated using Lequesne scale (0-3 points), and the histopathological changes of cartilage were observed under microscope after H.E. staining and the state of distribution of chondrocytes in different layers and the extracellular matrix was assessed using Mankin score (0-6 points). The contents of serum interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) were measured by using ELISA, and the expression levels of p-JAK2/JAK2, p-STAT3/STAT3 and MMP-9 in knee cartilage tissue were detected using Western blot. RESULTS: Compared with the blank group, the Lequesne score, Mankin score, and the contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression level of MMP-9 protein in knee cartilage tissue were significantly increased in the model group (P<0.01). In comparison with the model group, the Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression of MMP-9 protein in knee cartilage tissue were notably decreased in both the warm acupuncture and medication groups (P<0.01,P<0.05). The levels of Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 in knee cartilage tissue were significantly lower in the warm acupuncture group than in the medication group (P<0.01, P<0.05). No significant difference was found between the warm acupuncture and medication groups in the expression of MMP-9 protein (P>0.05). Outcomes of H.E. showed injury of the perichondrium of knee joint, obvious reduction of the cartilage matrix staining, cystic changes, clustered and disordered arrangement and severe pyknosis and necrosis of the surface cells with reduction of number of cells and increase of vacuoles in the model group, which was milder in both warm acupuncture and medication groups. CONCLUSION: Warm acupuncture can improve motor function and reduce cartilage injury in KOA rabbits, which may be related to its functions in inhibiting the secretion of pro-inflammatory factors and regulating JAK2/STAT3 signaling and downregulating MMP-9 expression in the cartilage tissue.

Ginsenoside Rb1 from Panax notoginseng Suppressed TNF-α-Induced Matrix Metalloproteinase-9 via the Suppression of Double-Strand RNA-Dependent Protein Kinase (PKR)/NF-κB Pathway.

Chronic inflammation is commonly accompanied by the stimulation of matrix metalloproteinases (MMPs) production and the degradation of the extracellular matrix. The overexpression of MMP-9 (Gelatinase B) highly participates in the progression of pathetic cardiac remodeling and liver cancer metastasis. Panax notoginseng (Burkill) F. H. Chen (Sanqi), a widely used traditional Chinese medicinal herb, shows myocardial protective and anti-tumor effects. In this study, we examined the inhibitory effect of different PNG extracts on tumor necrosis factor (TNF)-α-induced MMP-9 expression in cardiac myoblast H9c2 cells. Using a bioassay-guided fractionation scheme, the most active extract was fractionated by silica gel column chromatography and high-performance liquid chromatography until an active compound was obtained. The compound was identified as Ginsenoside Rb1 by nuclear magnetic resonance. Ginsenoside Rb1 inhibited TNF-α-induced MMP-9 production in both H9c2 and liver carcinoma HepG-2 cells. Interestingly, it did not affect the MMP-2 (Gelatinase A) level and the cell proliferation of the two cell lines. The inhibitory effects of Ginsenoside Rb1 may be due to its modulation of double-strand RNA-dependent protein kinase and nuclear factor kappa B signaling pathways. The results reveal the potential use of Ginsenoside Rb1 for the treatment of inflammatory and MMP-9-related cardiac remodeling and metastasis of hepatocellular carcinomas.

Capric Acid Behaves Agonistic Effect on Calcitriol to Control Inflammatory Mediators in Colon Cancer Cells.

Inflammation prompts cancer development and promotes all stages of tumorigenesis. Calcitriol is a nutraceutical essential regulator for host health benefits. However, the influence of calcitriol on inflammatory mediators involved in cancer cells is not clear. This study aimed to assess the sensitivity of calcitriol alone and combined with capric acid, and identify the possible influence of calcitriol on inflammatory mediators. The colorectal cancer cell line (HCT116) was induced by LPS/TNF-α and the inflammation and metastatic mediators (IL-1ß, IL-6, IL-17) were quantified in calcitriol and capric acid supplemented colon cancer cells. The mRNA and protein expression of MMP-2, NF-κB and COX-2 were quantified. The significant reduction in MMP-2 expression was confirmed at combination treatment by zymogram analysis. Our findings demonstrated the anti-inflammatory and anti-metastatic potentials of capric acid and calcitriol in individual exposure in a combination of human colon cancer cell lines (HCT116). These abilities may be due to the inhibition of COX-2 mediators and NF-κB transcription factor and reciprocally regulated MMP-2 and MMP-9 signaling pathways. These findings elucidate the activation of COX-2 and NF-κB via disruption of the cellular outer matrix could be considered a novel molecular target suitable for colorectal cancer therapy. This study confirmed that capric acid activates calcitriol sensitization in colon cancer cells and could be used as a successful supplement for intestinal diseases and colon aberrations.