Protective effect of Tao Hong Si Wu Decoction against inflammatory injury caused by intestinal flora disorders in an ischemic stroke mouse model.

BACKGROUND AND AIMS: Recent studies have shown that intestinal flora are involved in the pathological process of ischemic stroke (IS). The potential protective effect of the traditional Chinese prescription, Tao Hong Si Wu Decoction (THSWD), against inflammatory injury after IS and its underlying mechanisms of action were investigated in the current study. METHODS: Fifty SPF(Specefic pathogen Free) male C57 mice were randomly assigned to sham operation, model, THSWD low-dose (6.5 g/kg), medium-dose (13 g/kg) and high-dose (26 g/kg) groups (10 mice per group). Mouse models of transient middle cerebral artery occlusion were prepared via thread embolism. Neurological function score, hematoxylin-eosin (HE) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), 16S ribosomal DNA (rDNA) sequencing, quantitative reverse transcription PCR (qRT-PCR) and other methods were employed to elucidate the underlying molecular mechanisms. RESULTS: Notably, THSWD induced a reduction in the neurological function score (P < 0.01) and neuronal injury in brain tissue, increase in protein expression of Claudin-5 and zonula occludens-1 (ZO-1) in brain tissue(P < 0.01), and decrease in serum lipopolysaccharide (LPS)(P < 0.01), diamine oxidase (DAO)(P < 0.01) and D-lactic acid(P < 0.01, P < 0.05) levels to a significant extent. THSWD also inhibited the levels of tumor necrosis factor-α (TNF-α)(P < 0.01) and interleukin - 1ß (IL-1ß)(P < 0.01) in brain tissue, and increased alpha and beta diversity in ischemic stroke mice, along with a certain reversal effect on different microflora. Finally, THSWD inhibited the polarization of microglia cells(P < 0.01) and decreased the protein and gene expression of toll-like receptor-4 (TLR-4)(P < 0.01, P < 0.05) and nuclear factor kappa B (NF-κB)(P < 0.01) in brain tissue. CONCLUSION: Our data indicate that THSWD may interfere with inflammatory response in ischemic stroke by regulating intestinal flora and promoting intestinal barrier repair.

Analysis of global gene expression using RNA-sequencing reveals novel mechanism of Yanghe Pingchuan decoction in the treatment of asthma.

BACKGROUND: Yanghe Pingchuan decoction (YPD) has been used for asthma treatment for many years in China. We sought to understand the mechanism of YPD, and find more potential targets for YPD-based treatment of asthma. METHODS: An ovalbumin-induced asthma model in rats was created. Staining (hematoxylin and eosin, Masson) was used to evaluate the treatment effect of YPD. RNA-sequencing was carried out to analyze global gene expression, and differentially expressed genes (DEGs) were identified. Analysis of the functional enrichment of genes was done using the Gene Ontology database (GO). Analysis of signaling-pathway enrichment of genes was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Real-time reverse transcription-quantitative polymerase chain reaction was undertaken to measure expression of DEGs. RESULTS: Pathology showed that YPD had an improvement effect on rats with asthma. RNA-sequencing showed that YPD led to upregulated and downregulated expression of many genes. The YPD-based control of asthma pathogenesis may be related to calcium ion (Ca2+) binding, inorganic cation transmembrane transporter activity, microtubule motor activity, and control of canonical signaling (e.g., peroxisome proliferator-activated receptor, calcium, cyclic adenosine monophosphate). Enrichment analyses suggested that asthma pathogenesis may be related to Ca2 + binding and contraction of vascular smooth muscle. A validation experiment showed that YPD could reduce the Ca2 + concentration by inhibiting the Angiopoietin-II (Ang-II)/Phospholipase (PLA)/calmodulin (CaM0 signaling axis. CONCLUSION: Control of asthma pathogenesis by YPD may be related to inhibition of the Ang-II/PLA/CaM signaling axis, reduction of the Ca2+ concentration, and relaxation of airway smooth muscle (ASM).

Investigating the mechanism of action of Yanghe Pingchuan Granule in the treatment of bronchial asthma based on bioinformatics and experimental validation.

Background: Yanghe Pingchuan Granule (YPG) is a patented Chinese medicine developed independently by the Anhui Provincial Hospital of Traditional Chinese Medicine. For many years, it has been used for the treatment of asthma with remarkable clinical effects. However, the composition of YPG is complex, and its potential active ingredients and mechanism of action for the treatment of asthma are unknown. Materials and methods: In this study, we investigated the potential mechanism of action of YPG in the treatment of asthma through a combination of bioinformatics and in vivo experimental validation. We searched for active compounds in YPG and asthma targets from multiple databases and obtained common targets. Subsequently, a protein-protein interaction (PPI) network for compound disease was constructed using the protein interaction database for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, hematoxylin and eosin (H&E) staining, Masson staining, enzyme-linked immunosorbent assay (ELISA) analysis, immunofluorescence (IF) experiments, and Western blot (WB) experiments were performed to verify the possible mechanism of action of YPG for asthma treatment. Results: We obtained 72 active ingredients and 318 drug target genes that overlap with asthma. Serine/threonine-protein kinase (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF), interleukin (IL)-6, IL-1ß, vascular endothelial growth factor-A (VEGFA), prostaglandin-endoperoxide synthase 2 (PTGS2), caspase-3 (CASP3), mitogen-activated protein kinase 3 (MAPK3) and epidermal growth factor receptor (EGFR) were the most relevant genes in the PPI network. KEGG analysis showed a high number of genes enriched for the nuclear factor kappa-B (NF-κB) signaling pathway. Animal experiments confirmed that YPG reduced inflammatory cell infiltration and down-regulated the expression of ovalbumin-induced inflammatory factors. Furthermore, YPG treatment decreased the protein expression of NFĸB1, nuclear factor kappa B kinase subunit beta (IKBKB), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) in lung tissue. Conclusion: YPG has a positive effect on asthma by interfering with multiple targets. Furthermore, YPG may significantly inhibit the follicle-induced inflammatory response through the NF-ĸB signaling pathway.

Yanghe Pingchuan Granules Alleviate Airway Inflammation in Bronchial Asthma and Inhibit Pyroptosis by Blocking the TLR4/NF-κB/NRLP3 Signaling Pathway.

Bronchial asthma (BA) is a chronic inflammatory disease of the airway. Previous research has shown that Yanghe Pingchuan granules (YPGs) exert a precise therapeutic effect on BA. In our previous work, we showed that YPGs improved inflammation of the airways in rat models of BA. Other studies have shown that the pathogenesis of BA is closely related to pyroptosis and that the TOLL-like receptor pathway plays a key role in the mediation of pyroptosis. Therefore, in the present study, we established a rat model of BA by applying the concept of pyroptosis and used the TLR4/NF-κB/NRLP3 signaling pathway as the target and YPGs as the treatment method. We evaluated the effects of YPGs on airway inflammation and pyroptosis in the model rats by HE staining, Masson's staining, AP-PAS staining, western blotting, and real-time quantitative PCR. The results showed that Yanghe Pingchuan granules could significantly improve the inflammatory response of bronchial tissue in BA rats, reduce the content of inflammatory factors IL-1ß and IL-18, and inhibit the expression of pyroptosis factor. Meanwhile, YPG can block the TLR4/NF-κB signaling pathway. These findings suggest that YPG may be an effective drug for the treatment of BA by blocking the TLR4/NF-κB signaling pathway and inhibiting pyroptosis.

Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway.

Background: Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway. Materials and Methods: Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, ß-catenin, c-Myc, cyclin D1, GSK-3ß, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, ß-catenin, c-Myc, cyclin D1, GSK-3ß, and p-GSK-3ß (Ser9) protein were detected by immunofluorescence and Western blot. Results: We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b, ß-catenin, c-Myc, cyclin D1, and p-GSK-3ß/GSK-3ß mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway.

Naoluo Xintong Decoction in the Treatment of Ischemic Stroke: A Network Analysis of the Mechanism of Action.

The mechanism of action of Naoluo Xintong decoction (NLXTD) for the treatment of ischemic stroke (IS) is unknown. We used network analysis and molecular docking techniques to verify the potential mechanism of action of NLXTD in treating IS. The main active components of NLXTD were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and IS targets were collected from the Online Mendelian Inheritance in Man (OMIM), GeneCards, and Drugbank databases; their intersection was taken. In addition, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed and used to build protein-protein interaction networks. AutoDock Vina software was used for molecular docking, and animal experiments were conducted to verify the results. Hematoxylin and eosin staining was used to observe the brain morphology of rats in each group, and real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of relative mRNA in the brain tissue of rats. Western blot was used to detect the expression level of relative protein in the brain tissue of rats. Network analysis and molecular docking results showed that CASP3, NOS3, VEGFA, TNF, PTGS2, and TP53 are important potential targets for NLXTD in the treatment of IS. RT-qPCR and western blot results showed that NLXTD inhibited the expression of CASP3, TNF, PTGS2, and TP53 and promoted the expression of VEGFA and NOS3. NLXTD treats IS by modulating pathways and targets associated with inflammation and apoptosis in a multicomponent, multitarget manner.

Network pharmacology and experimental validation-based approach to understand the effect and mechanism of Taohong Siwu Decoction against ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (THSWD) is a classic prescription of traditional Chinese medicine that is mainly used for promoting blood circulation and alleviating blood stasis. THSWD is composed of Prunus persica (L.) Batsch, Carthamus tinctorius L., Ligusticum chuanxiong hort, Angelica sinensis (Oliv.) Diels, Rehmannia glutinosa (Gaertn.) DC, and Paeoniae Radix Alba. This prescription eliminates blood stasis, supplements blood, and dredges the body as an auxiliary treatment. AIM OF THE STUDY: To investigate the mechanistic effects of THSWD in the treatment of cerebral ischemia. MATERIALS AND METHODS: we downloaded 39 blood components for THSWD from the PharmMapper database for target prediction studies and identified the targets of cerebral ischemia. We identified the intersection between the components and targets, constructed a protein-protein interaction (PPI) network, carried out GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. a rat model of cerebral ischemia was established in rats, and the results of network pharmacology were verified by in vivo experiments. RESULTS: Established a component-target-pathway network, further transcriptomics analysis identified a total of 11 target genes (Plau, Fabp4, Mmp9, Mmp12, Cfd, Lcn2, Trem1, Lgals3, Hmox1, Selp and Slc6a4), a total of seven pathways (focal adhesion, complement and coagulation cascades, Staphylococcus aureus infection, malaria, transcriptional dysregulation in cancer, progesterone-mediated oocyte maturation, and the PI3K-Akt signaling pathway), because both targets genes and the complement and coagulation cascade signaling pathways mediate inflammatory responses, the signaling pathways associated with the complement and coagulation cascades were selected for experimental verification. We detected inflammatory factors and several key proteins in the complement and coagulation cascade signaling pathway (C1qb, C1qc, C3ar1, C5ar1, and Cfd). Analysis showed that THSWD can reduce the release of inflammatory factors and inhibit activation of the complement signaling pathways, thereby protecting against ischemic stroke disease. CONCLUSIONS: Our findings provide preliminary clarification of the predominant mechanism of action of THSWD when used to treat ischemic stroke.

Exploration of the Potential Mechanism of Tao Hong Si Wu Decoction for the Treatment of Breast Cancer Based on Network Pharmacology and In Vitro Experimental Verification.

BACKGROUND: Tao Hong Si Wu Decoction (THSWD) is a well-known traditional Chinese medicine used clinically alone or combined with drugs to treat breast cancer. However, there has been no study to date on the underlying mechanisms of its therapeutic effects. OBJECTIVES: To explore the potential mechanism of THSWD for the treatment of breast cancer using network pharmacology and experimental research. METHODS: The active ingredients of THSWD were screened according to Lipinski's rule of five based on the 107 ingredients of THSWD identified by UPLC-Q-TOF-MSE. The targets of THSWD and breast cancer from multiple databases were collected, and a Compound-Target-Pathway network based on protein-protein interaction (PPI) was constructed. Gene ontology (GO) analysis and KEGG pathway analysis were performed via the DAVID server. Molecular docking studies verified the selected key ingredients and key targets. The results of network pharmacology were verified by in vitro experiments. Including the effects of THSWD drug-containing rat serum (THSWD serum) on cell proliferation, and on the targets HRAS, MAPK1, AKT1, GRB2, and MAPK14 were assayed by RT-qPCR and Western blot assays. RESULTS: In total, 27 active ingredients including 8 core components, were obtained from 107 ingredients and 218 THSWD target genes for the treatment of breast cancer were identified. THSWD is active in the treatment of breast cancer by targeting Ras, FoxO, PI3K-Akt and other signaling pathways. MCF-7 and MDA-MB-231 cell proliferation was inhibited by THSWD serum in a time and concentration dependent manner. THSWD could regulated the RNA and protein expression of core targets HRAS, MAPK1, AKT1, GRB2, and MAPK14 for treatment of breast cancer. CONCLUSION: The results of network pharmacology study showed that THSWD is active against breast cancer by intervening with multiple targets and pathways. Luteolin, kaempferol, senkyunolide E, and other 8 compounds may be the core active ingredients of THSWD in the treatment of breast cancer. THSWD treatment of breast cancer may be related to targeting Ras, FoxO, PI3K-Akt, and other signal pathways associated with the core targets HRAS, MAPK1, AKT1, GRB2, and MAPK14.

Analysis of the active components and metabolites of Taohong Siwu decoction by using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Taohong Siwu Decoction is a classic Chinese medicine prescription for treatment of cerebral ischemia and gynecological diseases. However, the active ingredients of Taohong Siwu Decoction have not been identified. In this study, a ultra performance liquid chromatography quadrupole time-of-flight mass system was used to analyze the active components and metabolites of Taohong Siwu Decoction absorbed into the blood and the brain. A total of 39 active compounds and 90 metabolites were identified in the blood and brain by comparing retention times, accurate masses, fragmentation patterns, and literature data. The results showed that flavonoids (Carthamus tinctorius L), aromatic organic acids, and benzoquinones (Angelica sinensis (Oliv.) Diels and Ligusticumchuanxiong hort) were prominent active ingredients in Taohong Siwu Decoction. Furthermore, hydrolysis, glucuronidation, and sulfation were identified as the main metabolic pathways of Taohong Siwu Decoction in vivo. This was the first study to characterize the active components and metabolites of Taohong Siwu Decoction in the blood and brain using ultra performance liquid chromatography quadrupole time-of-flight mass.

Bupleurum polysaccharides ameliorated renal injury in diabetic mice associated with suppression of HMGB1-TLR4 signaling.

Bupleurum polysaccharides (BPs) is isolated from Bupleurum smithii var. parvifolium, a key traditional Chinese medicine. The study was to investigate the effects of BPs on diabetic kidney injury. After two intraperitoneal injections of streptozotozin (STZ) 100 mg·kg-1, renal injury in diabetic mice was induced and BPs was orally administrated at dosages of 30 and 60 mg·kg-1·d-1. The STZ injected mice developed renal function damage, renal inflammation and fibrosis known as diabetic kidney disease (DKD). BPs significantly reduced serum creatinine level and urinary albumin excretion rate, with the attenuated swelling of kidneys. BPs treatment obviously alleviated the pathological damage of renal tissue. The progression of renal injury in BPs treated mice was inhibited with less expression of type IV collagen (Col IV), fibronectin (FN) and α-smooth muscle actin (α-SMA). The inhibition of inflammation in kidney was associated with the reduced level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). BPs administration suppressed the over-expression of toll like receptor 4 (TLR4) and high-mobility group box 1 (HMGB1) with lowered activity of nuclear factor kappa B (NF-κB) in renal tissue of diabetic mice. Oral administration of BPs effectively prevented the development ofrenal injury in diabetic mice. This study suggested that the protection provided by BPs might affect through the interruption of HMGB1-TLR4 pathway, leading to the inhibition of renal inflammation and fibrotic process.

UPLC-Q-TOF-MS Study of the Mechanism of THSWD for Breast Cancer Treatment.

Taohong Siwu decoction (THSWD) is a classic traditional Chinese medicine (TCM) prescription that is widely used in the clinical treatment of gynecological and cerebrovascular diseases. Here we used a method that coupled ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in which both positive and negative ion modes were established to investigate the major constituents in THSWD. A Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was used to separate the aqueous extract of THSWD. The mobile phase consisted of 0.1% aqueous formic acid (A) and acetonitrile (B). Ninety-five components were identified in two different ion modes, including aromatic acids, flavones, polysaccharides, volatile oils monoterpene glycosides, aromatic cyanogenic glycosides, and others. Pathological changes in tumors and serum expression of interleukin-4 in a mouse model of breast cancer were detected after THSWD treatment. The results showed that THSWD had obvious therapeutic effects. This study establishes a material basis for the use of THSWD in the treatment of breast cancer.

Houttuyniacordata Thunb. polysaccharides ameliorates lipopolysaccharide-induced acute lung injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata (HC) has been used as a folk therapy to treat pulmonary infections. This study aimed to determine the role and mechanism of action of polysaccharides isolated from HC (HCP) in lipopolysaccharide (LPS)-induced ALI in the mice. MATERIALS AND METHODS: LPS was delivered by the intratracheal route to Balb/c mice 2h before HCP (40, 80 and 160mg/kg) administration. RESULTS: The number of total cells, protein and tumor necrosis factor-α (TNF-α) concentrations in bronchoalveolar lavage fluid, the wet/dry weight ratio (w/d) of lungs and pulmonary pathology of each mouse were analyzed, it was found that HCP significantly alleviated ALI induced by LPS. Moreover, in lungs of mice, it was found that the infiltration of inflammatory cells, the expression of Toll-like receptor 4 and complement deposition were significantly decreased by HCP treatment. In vitro assays showed that C5a, a complement activation product, induced significant macrophage migration and treatment with HCP prevented it. The in vitro results also proved that LPS increased nitric oxide and pro-inflammatory cytokines (TNF-α, interleukin-6, and interleukin-1ß) production, and HCP antagonized these effects of LPS. It was also found that HCP alone augmented secretion of some pro-inflammatory cytokines. CONCLUSION: These results indicate that HCP may alleviate LPS induced lung inflammatory injury, which may be associated with its inhibitory effect on the over activation of complement and macrophages. This suggests a potential role to treat ALI.

Therapeutic Effects of Bupleurum Polysaccharides in Streptozotocin Induced Diabetic Mice.

Diabetes mellitus is related to low-grade chronic inflammation and oxidative stress. Bupleurum Polysaccharides (BPs), isolated from Bupleurum smithii var. parvifolium has anti-inflammatory and anti-oxidative properties. However, little is known about its therapeutic effects on diabetes. In this experiment, the effects of BPs on alleviation of diabetes and the underlying mechanisms were investigated. Diabetic mice model was established via successive intraperitoneal injections of streptozotocin (100 mg/kg body weight) for two days. Mice with blood glucose levels higher than 16.8mmol/L were selected for experiments. The diabetic mice were orally administered with BPs (30 and 60 mg/kg) once a day for 35 days. BPs not only significantly decreased levels of blood glucose, but also increased those of serum insulin and liver glycogen in diabetic mice compared to model mice. Additionally, BPs adminstration improved the insulin expression and suppressed the apoptosis in pancreas of the diabetic mice. Histopathological observations further demonstrated that BPs protected the pancreas and liver from oxidative and inflammatory damages. These results suggest that BPs protect pancreatic ß cells and liver hepatocytes and ameliorate diabetes, which is associated with its anti-oxidative and anti-inflammatory properties.