Integrating metabolomics and network pharmacology analysis to explore mechanism of Pueraria lobata against pulmonary fibrosis: Involvement of arginine metabolism pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi is a typical medicinal and edible plant with a long application history in China and Southeast Asia. As a widely used traditional medicine, P. lobata exhibits the properties of anti-inflammatory, antipyretic, antioxidant, relieving cough and asthma. Particularly, the increasing evidence indicates that the P. lobata has the therapeutic effect on fibrotic-related diseases in terms of metabolic regulation. However, the mechanisms of P. lobata on pulmonary fibrosis (PF) has not been thoroughly explored. AIM OF THE STUDY: This study aimed to explore the effect of arginine metabolites of P. lobata against PF model by integrating metabolomics and network pharmacology analysis. It might provide a new idea for the target finding of P. lobata anti-pulmonary fibrosis. MATERIALS AND METHODS: In this study, the Sprague Dawley (SD) rats were randomly divided into five experimental groups: saline-treated control group, bleomycin-induced fibrosis group, prednisolone acetate group, P. lobata 3.2 g/kg group and P. lobata 6.4 g/kg group. The therapeutic effect of P. lobata on bleomycin-induced PF in rats was evaluated by clinical symptoms such as lung function, body weight, hematoxylin eosin staining (HE), Masson staining and hydroxyproline assay. Next, the plasma metabolomics analysis was carried out by LC-MS to explore the pathological differences between the group of control, PF and P. lobata-treated rats. Then, the network pharmacology study coupled with experimental validation was conducted to analysis the results of metabolic research. We constructed the "component-target-disease" network of P. lobata in the treatment of PF. In addition, the molecular docking method was used to verify the interaction between potential active ingredients and core targets of P. lobata. Finally, we tested NOS2 and L-OT in arginine-related metabolic pathway in plasma of the rats by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was performed to observe the level of TNF-α mRNA and MMP9 mRNA. And we tested the expression of TNF-α and MMP9 by Western blot analysis. RESULTS: Our findings revealed that P. lobata improved lung function and ameliorated the pathological symptoms, such as pathological damage, collagen deposition, and body weight loss in PF rats. Otherwise, the plasma metabolomics were employed to screen the differential metabolites of amino acids, lipids, flavonoids, arachidonic acid metabolites, glycoside, etc. Finally, we found that the arginine metabolism signaling mainly involved in the regulating of P. lobata on the treatment of PF rats. Furtherly, the network pharmacology predicted that the arginine metabolism pathway was contained in the top 20 pathways. Next, we integrated metabolomics and network pharmacology that identified NOS2, MMP9 and TNF-α as the P. lobata regulated hub genes by molecular docking. Importantly, it indicated a strong affinity between the puerarin and the NOS2. P. lobata attenuated TNF-α, MMP-9 and NOS2 levels, suppressed TNF-α and MMP-9 protein expression, and decreased L-OT and NOS2 content in PF rats. These results indicated that the effects of P. lobata may ameliorated PF via the arginine metabolism pathway in rats. Therefore, P. lobata may be a potential therapeutic agent to ameliorated PF. CONCLUSION: In this work, we used metabolomics and network pharmacology to explore the mechanisms of P. lobata in the treatment of PF. Finally, we confirmed that P. lobata alleviated BLM-induced PF in rats by regulating arginine metabolism pathway based on reducing the L-OT and NOS2-related signal molecular. The search for the biomarkers finding of arginine metabolism pathway revealed a new strategy for P. lobata in the treatment of PF.

Manual acupuncture ameliorates inflammatory pain by upregulating adenosine A3 receptor in complete Freund's adjuvant-induced arthritis rats.

BACKGROUND: Adenosine A3 receptor (A3R) exerts analgesic, anti-inflammatory, and anti-nociceptive effects. In this study, we determined the analgesic mechanism of manual acupuncture (MA) in rats with complete Freund's adjuvant (CFA)-induced arthritis and explored whether MA ameliorates inflammation in these rats by upregulating A3R. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into the following groups: Control, CFA, CFA + MA, CFA + sham MA, CFA + MA + DMSO, CFA + MA + IB-MECA, and CFA + MA + Reversine groups. The arthritis rat model was induced by injecting CFA into the left ankle joints. Thereafter, the rats were subjected to MA (ST36 acupoint) for 3 days. The clinical indicators paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and open field test (OFT) were used to determine the analgesic effect of MA. In addition, to explore the effect of A3R on inflammation after subjecting arthritis rats to MA, IB-MECA (A3R agonist) and Reversine (A3R antagonist) were injected into ST36 before MA. RESULTS: MA ameliorated the pathological symptoms of CFA-induced arthritis, including the pain indicators PWL and PWT, number of rearing, total ambulatory distance, and activity trajectory. Furthermore, after MA, the mRNA and protein expression of A3R was upregulated in CFA-induced arthritis rats. In contrast, the protein levels of TNF-α, IL-1ß, Rap1, and p-p65 were downregulated after MA. Interestingly, the A3R agonist and antagonist further downregulated and upregulated inflammatory cytokine expression, respectively, after MA. Furthermore, the A3R antagonist increased the degree of ankle swelling after MA. CONCLUSION: MA can alleviate inflammatory pain by inhibiting the NF-κB signaling pathway via upregulating A3R expression of the superficial fascia of the ST36 acupoint site in CFA-induced arthritis rats.

Artemisinin analog SM934 alleviates epithelial barrier dysfunction via inhibiting apoptosis and caspase-1-mediated pyroptosis in experimental colitis.

Intestinal barrier disruption due to the intestinal epithelial cells' (IECs) death is one of the critical pathological features of inflammatory bowel diseases (IBDs). SM934, an artemisinin analog, has previously been proven to ameliorate colitis induced by dextran sulfate sodium (DSS) in mice by suppressing inflammation response. In this study, we investigated the protective effects of SM934 on the epithelial barrier and the underlying mechanism in trinitrobenzene sulfonic acid (TNBS)-induced colitis mice. We demonstrated that SM934 restored the body weight and colon length, and improved the intestine pathology. Furthermore, SM934 treatment preserved the intestinal barrier function via decreasing the intestinal permeability, maintaining epithelial tight junction (TJ) protein expressions, and preventing apoptosis of epithelial cells, which were observed both in the colon tissue and the tumor necrosis factor-α (TNF-α)-induced human colonic epithelial cell line HT-29. Specifically, SM934 reduced the pyroptosis of IECs exposed to pathogenic signaling and inhibited pyroptosis-related factors such as NOD-like receptor family pyrin domain containing 3 (NLRP3), adapter apoptosis-associated speck-like protein (ASC), cysteine protease-1 (caspase-1), gasdermin (GSDMD), interleukin-18 (IL-18), and high-mobility group box 1 (HMGB1) both in colon tissue and lipopolysaccharide (LPS) and adenosine triphosphate (ATP) co-stimulated HT-29 cells in vitro. Moreover, SM934 interdicted pyroptosis via blocking the transduction of mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways. In conclusion, SM934 protected TNBS-induced colitis against intestinal barrier disruption by inhibiting the apoptosis and pyroptosis of epithelial cells via the NLRP3/NF-κB/MAPK signal axis, and intestinal barrier protection in company with an anti-inflammatory strategy might yield greater benefits in IBD treatment.

The artemisinin analog SM934 alleviates dry eye disease in rodent models by regulating TLR4/NF-κB/NLRP3 signaling.

Dry eye disease (DED) is a multifactorial disorder of the tears and ocular surface characterized by manifestations of dryness and irritation. Although the pathogenesis is not fully illuminated, it is recognized that inflammation has a prominent role in the development and deterioration of DED. ß-aminoarteether maleate (SM934) is a water-soluble artemisinin derivative with anti-inflammatory and immunosuppressive activities. In this study, we established scopolamine hydrobromide (SCOP)-induced rodent model as well as benzalkonium chloride (BAC)-induced rat model to investigate the therapeutic potential of SM934 for DED. We showed that topical application of SM934 (0.1%, 0.5%) significantly increased tear secretion, maintained the number of conjunctival goblet cells, reduced corneal damage, and decreased the levels of inflammatory mediators (TNF-α, IL-6, IL-10, or IL-1ß) in conjunctiva in SCOP-induced and BAC-induced DED models. Moreover, SM934 treatment reduced the accumulation of TLR4-expressing macrophages in conjunctiva, and suppressed the expression of inflammasome components, i.e., myeloid differentiation factor88 (MyD88), Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), and cleaved caspase 1. In LPS-treated RAW 264.7 cells, we demonstrated that pretreatment with SM934 (10 µM) impeded the upregulation of TLR4 and downstream NF-κB/NLRP3 signaling proteins. Collectively, artemisinin analog SM934 exerts therapeutic benefits on DED by simultaneously reserving the structural integrity of ocular surface and preventing the corneal and conjunctival inflammation, suggested a further application of SM934 in ophthalmic therapy, especially for DED.

[Application of active components from traditional Chinese medicine in treatment of inflammatory bowel disease].

Inflammatory bowel disease(IBD) is a non-specific and chronic recurrent autoimmune disease that involves the gastrointestinal tract. Clinical symptoms of intestinal bleeding, diarrhea, and weight loss threat to human health and induce colorectal cancer. The pathogenesis included living environment, genetic factors, immune cell infiltration and immune stress, weakened mucosal barrier defense and intestinal flora imbalance. At present, clinical treatment drugs mainly include aminosalicylic acid, corticosteroids, immunosuppressants, biological agents, etc., in view of the disadvantages of poor therapeutic effect and expensive price. The active ingredients of traditional Chinese medicine(TCM) in the treatment IBD have various biological activities and multiple targets such as anti-inflammatory, antibacterial, anti-tumor and immune regulation. This article summarized the application and the research progress in protecting intestinal epithelial barrier, maintaining intestinal microbial homeostasis, inhibiting causative factors, and regulating Th1/Th17/Treg balance about TCM in the treatment of IBD. The review provided new ideas for further development of the new drugs on the mechanism based on active ingredients of TCM in IBD treatment.

Artemisinin analogue SM934 ameliorates DSS-induced mouse ulcerative colitis via suppressing neutrophils and macrophages.

Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease (IBD) characterized by complicated and relapsing inflammation in the gastrointestinal tract. SM934 is a water-soluble artemisinin analogue that shows anti-inflammatory and immuno-regulatory effects. In this study, we investigated the effects of SM934 on UC both in vivo and in vitro. A mouse model of colitis was established in mice by oral administration of 5% dextran sulfate sodium (DSS). SM934 (3, 10 mg/kg per day, ig) was administered to the mice for 10 days. After the mice were sacrificed, colons, spleens and mesenteric lymph nodes (MLNs) were collected for analyses. We showed that SM934 administration restored DSS-induced body weight loss, colon shortening, injury and inflammation scores. Furthermore, SM934 administration significantly decreased the disease activity index (DAI), histopathological scores, and myeloperoxidase (MPO) activities in colonic tissues. Moreover, SM934 administration dose-dependently decreased the mRNA and protein levels of DSS-induced pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α), and the percentage of macrophages and neutrophils in colon tissues. The effects of SM934 on LPS-stimulated RAW 264.7 cells and THP-1-derived macrophages were examined in vitro. Treatment with SM934 (0.8, 8, 80 µmol/L) dose-dependently decreased the production of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells and THP-1-derived macrophages via inhibiting activation of the NF-κB signaling. Our results reveal the protective effects of SM934 on DSS-induced colitis can be attributed to its suppressing effects on neutrophils and macrophages and its inhibitory role in the NF-κB signaling, suggests that SM934 might be a potential effective drug for ulcerative colitis.

(5R)-5-hydroxytriptolide ameliorates anti-glomerular basement membrane glomerulonephritis in NZW mice by regulating Fcγ receptor signaling.

(5R)-5-hydroxytriptolide (LLDT-8) is a novel triptolide analog that has been identified as a promising candidate for treating autoimmune diseases and has been shown to be effective in treating murine collagen-induced arthritis and lupus nephritis. In the present study, we investigated the therapeutic effect and possible mechanism of action of LLDT-8 in a murine anti-glomerular basement membrane (GBM) glomerulonephritis model. NZW mice were injected with rabbit anti-GBM serum (500 µL, ip). The mice were orally treated with LLDT-8 (0.125 mg/kg, every other day) or a positive control prednisolone (2 mg/kg every day) for 14 d. Blood and urine samples as well as spleen and kidney tissues were collected for analyses. LLDT-8 treatment did not affect the generation of mouse anti-rabbit antibodies. LLDT-8 significantly reversed established proteinuria, improved renal histopathology and attenuated renal dysfunction in glomerulonephritis mice. Furthermore, LLDT-8 inhibited inflammation in the kidney evidenced by significantly decreasing C3 and IgG deposition, reducing the levels of the pathogenic cytokines TNF-α, IL-6, IL-17, and IFN-γ, and reducing related chemokine expression and leukocyte infiltration in kidneys. Moreover, LLDT-8 treatment significantly increased the expression of FcγRIIB in the kidney and spleen. In addition, the treatment restored the reduced expression of FcγRIIB on the surface of kidney effector cells, CD11b+ cells, and interfered with FcγR-dependent signaling, especially FcγRIIB-mediated downstream kinases, such as BTK. These results demonstrate that LLDT-8 ameliorates anti-GBM glomerulonephritis by regulating the Fcγ receptor signaling.

Aesculetin (6,7-dihydroxycoumarin) exhibits potent and selective antitumor activity in human acute myeloid leukemia cells (THP-1) via induction of mitochondrial mediated apoptosis and cancer cell migration inhibition.

PURPOSE: The main target of the present research was to examine the antitumor properties of aesculetin in human acute myeloid leukemia cancer cells (THP-1) and peripheral blood mono-nucleated cells (PBMCs) (used as normal cell line model) along with the determination of its effects on induction of apoptosis, inhibition of cancer cell migration and changes in Bcl-2/Bax protein expressions. METHODS: MTT colorimetric bioassay was performed to study the impact of this natural compound on cytotoxicity of both cell types. Moreover, transmission electron microscopy (TEM), inverted phase contrast and fluorescence microscopic techniques were used to study the effects on cell morphology and cellular ultrastructural details connected with apoptosis. The effects of aesculetin on Bcl-2/Bax protein expressions were assessed by Western blot method. RESULTS: Selective and dose-dependent antiproliferative activity of aesculetin in human acute myeloid leukemia cancer cells was observed. However, the compound did not induce significant cell growth inhibition of PBMCs, which were used as normal cell controls. Fluorescence and inverted phase contrast microscopic techniques revealed that aesculetin led to morphological changes suggestive of apoptosis (cell shrinkage, chromatin abridgment and membrane blebbing). TEM analysis showed that aesculetin led to fragmented plasma membrane along with appearance of spherical projections (apoptotic bodies). The wound scratch widened after aesculetin treatment, indicating that aesculetin exhibits anticancer effects by suppressing the cancer cell migration. Aesculetin led to significant and dose-dependent reduction in the Bcl-2 expression while the expression of Bax was significantly enhanced resulting in overall reduction of Bcl-2/Bax ratio. CONCLUSION: The results of the present work revealed that aesculetin exhibits selective anticancer effects in THP-1 human leukemia cells without causing much cytotoxicity in PBMCs. It also led to significant apoptosis induction, inhibition of cancer cell migration and decrease in Blc-2/Bax ratio.