Exploring the immune-inflammatory mechanism of Maxing Shigan Decoction in treating influenza virus A-induced pneumonia based on an integrated strategy of single-cell transcriptomics and systems biology.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza virus. Maxing Shigan Decoction (MXSGD) is a commonly used traditional Chinese medicine prescription for the prevention and treatment of influenza. However, its mechanism remains unclear. METHOD: The mice model of influenza A virus pneumonia was established by nasal inoculation. After 3 days of intervention, the lung index was calculated, and the pathological changes of lung tissue were detected by HE staining. Firstly, transcriptomics technology was used to analyze the differential genes and important pathways in mouse lung tissue regulated by MXSGD. Then, real-time fluorescent quantitative PCR (RT-PCR) was used to verify the changes in mRNA expression in lung tissues. Finally, intestinal microbiome and intestinal metabolomics were performed to explore the effect of MXSGD on gut microbiota. RESULTS: The lung inflammatory cell infiltration in the MXSGD group was significantly reduced (p < 0.05). The results of bioinformatics analysis for transcriptomics results show that these genes are mainly involved in inflammatory factors and inflammation-related signal pathways mediated inflammation biological modules, etc. Intestinal microbiome showed that the intestinal flora Actinobacteriota level and Desulfobacterota level increased in MXSGD group, while Planctomycetota in MXSGD group decreased. Metabolites were mainly involved in primary bile acid biosynthesis, thiamine metabolism, etc. This suggests that MXSGD has a microbial-gut-lung axis regulation effect on mice with influenza A virus pneumonia. CONCLUSION: MXSGD may play an anti-inflammatory and immunoregulatory role by regulating intestinal microbiome and intestinal metabolic small molecules, and ultimately play a role in the treatment of influenza A virus pneumonia.

Dan-shen Yin promotes bile acid metabolism and excretion to prevent atherosclerosis via activating FXR/BSEP signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-shen Yin (DSY), a traditional prescription, has been demonstrated to be effective in decreasing hyperlipidemia and preventing atherosclerosis (AS), but its mechanism remains unknown. We hypothesized that DSY activates farnesoid X receptor (FXR) to promote bile acid metabolism and excretion, thereby alleviating AS. AIM OF THE STUDY: This study was designed to explore whether DSY reduces liver lipid accumulation and prevents AS by activating FXR and increasing cholesterol metabolism and bile acid excretion. MATERIALS AND METHODS: The comprehensive chemical characterization of DSY was analyzed by UHPLC-MS/MS. The AS models of ApoE-/- mice and SD rats was established by high-fat diet and high-fat diet combined with intraperitoneal injection of vitamin D3, respectively. The aortic plaque and pathological changes were used to evaluate AS. Lipid levels, H&E staining and oil red O staining were used to evaluate liver lipid accumulation. The cholesterol metabolism and bile acid excretion were evaluated by enzyme-linked immunosorbent assay, UPLC-QQQ/MS. In vitro, the lipid and FXR/bile salt export pump (BSEP) levels were evaluated by oil red O staining, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. RESULTS: A total of 36 ingredients in DSY were identified by UPLC-MS/MS analysis. In vivo, high-dose DSY significantly inhibited aortic intimal thickening, improved arrangement disorder, tortuosity, and rupture of elastic fibers, decreased lipid levels, and reduced the number of fat vacuoles and lipid droplets in liver tissue in SD rats and ApoE-/- mice. Further studies found that high-dose DSY significantly reduced liver lipid and total bile acids levels, increased liver ursodeoxycholic acid (UDCA) and other non-conjugated bile acids levels, increased fecal total cholesterol (TC) levels, and augmented FXR, BSEP, cholesterol 7-alpha hydroxylase (CYP7A1), ATP binding cassette subfamily G5/G8 (ABCG5/8) expression levels, while decreasing ASBT expression levels. In vitro studies showed that DSY significantly reduced TC and TG levels, as well as lipid droplets, while also increasing the expression of ABCG5/8, FXR, and BSEP in both HepG2 and Nr1h4 knockdown HepG2 cells. CONCLUSION: This study demonstrated that DSY promotes bile acid metabolism and excretion to prevent AS by activating FXR. For the prevent of AS and drug discovery provided experimental basis.

Integrative analysis of network pharmacology and proteomics reveal the protective effect of Xiaoqinglong Decotion on neutrophilic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoqinglong Decotion (XQLD) is a commonly used Chinese herbal formula in clinical practice, especially for allergic diseases such as asthma. However, its intrinsic mechanism for the treatment of neutrophilic asthma (NA) remains unclear. AIM OF THE STUDY: The aim of this study was to evaluate the efficacy and potential mechanisms of XQLD on NA using network pharmacology and in vivo experiments. MATERIALS AND METHODS: First, the active compounds, potential targets and mechanisms of XQLD against NA were initially elucidated by network pharmacology. Then, OVA/CFA-induced NA mice were treated with XQLD to assess its efficacy. Proteins were then analyzed and quantified using a Tandem Mass Tags approach for differentially expressed proteins (DEPs) to further reveal the mechanisms of NA treatment by XQLD. Finally, the hub genes, critical DEPs and potential pathways were validated. RESULTS: 176 active compounds and 180 targets against NA were identified in XQLD. Protein-protein interaction (PPI) network revealed CXCL10, CX3CR1, TLR7, NCF1 and FABP4 as hub genes. In vivo experiments showed that XQLD attenuated inflammatory infiltrates, airway mucus secretion and remodeling in the lungs of NA mice. Moreover, XQLD significantly alleviated airway neutrophil inflammation in NA mice by decreasing the expression of IL-8, MPO and NE. XQLD also reduced the levels of CXCL10, CX3CR1, TLR7, NCF1 and FABP4, which are closely associated with neutrophil inflammation. Proteomics analysis identified 28 overlapping DEPs in the control, NA and XQLD groups, and we found that XQLD inhibited ferroptosis signal pathway (elevated GPX4 and decreased ASCL3) as well as the expression of ARG1, MMP12 and SPP1, while activating the Rap1 signaling pathway. CONCLUSION: This study revealed that inhibition of ARG1, MMP12 and SPP1 expression as well as ferroptosis pathways, and activation of the Rap1 signaling pathway contribute to the therapeutic effect of XQLD on NA.

Exploring the mechanism of Corbrin capsules in the intervention of AKI-COVID-19 based on network pharmacology combined with GEO dataset.

AIM: of the study: This study used network pharmacology and the Gene Expression Omnibus (GEO) database to investigate the therapeutic effects of Corbrin capsules on acute kidney injury (AKI)-COVID-19 (coronavirus disease 2019). MATERIALS AND METHODS: The active constituents and specific molecular targets of Corbrin capsules were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Swiss Target Prediction databases. The targets related to AKI and COVID-19 disease were obtained from the Online Mendelian Inheritance in Man (OMIM), GeneCards, and GEO databases. A protein-protein interaction (PPI) network was constructed by utilizing Cytoscape. To enhance the analysis of pathways associated with the pathogenesis of AKI-COVID-19, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Furthermore, immune infiltration analysis was performed by using single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT. Molecular docking was used to assess interactions between differentially expressed genes and active ingredients. Verification was performed by utilizing GEO databases and in vivo assays. RESULTS: This study revealed an overlap of 18 significantly differentially expressed genes between the Corbrin capsules group and the AKI-COVID-19 target group. Analysis of the PPI network identified TP53, JAK2, PIK3CA, PTGS2, KEAP1, and MCL1 as the top six core protein targets with the highest degrees. The results obtained from GO and KEGG analyses demonstrated that the target genes were primarily enriched in the apoptosis and JAK-STAT signaling pathways. Moreover, the analysis of immune infiltration revealed a notable disparity in the percentage of quiescent memory CD4 + T cells. Western blot analyses provided compelling evidence suggesting that the dysregulation of 6 core protein targets could be effectively reversed by Corbrin capsules. CONCLUSION: This study revealed the key components, targets, and pathways involved in treating AKI-related COVID-19 using Corbrin capsules. This study also provided a new understanding of the molecular mechanisms underlying this treatment.

Kidney-tonifying blood-activating decoction delays ventricular remodeling in rats with chronic heart failure by regulating gut microbiota and metabolites and p38 mitogen-activated protein kinase/p65 nuclear factor kappa-B/aquaporin-4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Myocardial infarction has likely contributed to the increased prevalence of heart failure(HF).As a result of ventricular remodeling and reduced cardiac function, colonic blood flow decreases, causing mucosal ischemia and hypoxia of the villous structure of the intestinal wall.This damage in gut barrier function increases bowel wall permeability, leading to fluid metabolism disorder,gut microbial dysbiosis, increased gut bacteria translocation into the circulatory system and increased circulating endotoxins, thus promoting a typical inflammatory state.Traditional Chinese Medicine plays a key role in the prevention and treatment of HF.Kidney-tonifying Blood-activating(KTBA) decoction has been proved for clinical treatment of chronic HF.However,the mechanism of KTBA decoction on chronic HF is still unclear. AIMS OF THE STUDY: The effect of KTBA decoction on gut microbiota and metabolites and p38MAPK/p65NF-κB/AQP4 signaling in rat colon was studied to investigate the mechanism that KTBA decoction delays ventricular remodeling and regulates water metabolism disorder in rats with HF after myocardial infarction based on the theory of "Kidney Storing Essence and Conducting Water". MATERIAL AND METHODS: In vivo,a rat model of HF after myocardial infarction was prepared by ligating the left anterior descending coronary artery combined with exhaustive swimming and starvation.The successful modeling rats were randomly divided into five groups:model group, tolvaptan group(gavaged 1.35mg/(kg•D) tolvaptan),KTBA decoction group(gavaged 15.75g/(kg•D) of KTBA decoction),KTBA decoction combined with SB203580(p38MAPK inhibitor) group(gavaged 15.75g/(kg•D) of KTBA decoction and intraperitoneally injected 1.5mg/(kg•D) of SB203580),and KTBA decoction combined with PDTC(p65NF-kB inhibitor) group(gavaged 15.75g/(kg•D) of KTBA decoction and intraperitoneally injected 120mg/(kg•D) of PDTC).The sham-operation group and model group were gavaged equal volume of normal saline.After 4 weeks of intervention with KTBA decoction,the effect of KTBA decoction on the cardiac structure and function of chronic HF model rats was observed by ultrasonic cardiogram.General state and cardiac index in rats were evaluated.Enzyme linked immunosorbent assay(ELISA) was used to measure N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration in rat serum.Hematoxylin and eosin(H&E) staining,and transmission electron microscope(TEM) were used to observe the morphology and ultrastructure of myocardial and colonic tissue,and myocardial fibrosis was measured by Masson's staining.Cardiac E-cadherin level was detected by Western blot.The mRNA expression and protein expression levels of p38MAPK,I-κBα, p65NF-κB,AQP4,Occludin and ZO-1 in colonic tissue were detected by reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR) and immunohistochemistry. Protein expression of p38MAPK, p-p38MAPK,I-κBα,p-I-κBα,p65NF-κB, p-p65NF-κB,AQP4,Occludin and ZO-1 in rat colon was detected using Western blot.Colonic microbiota and serum metabolites were respectively analyzed by amplicon sequencing and liquid chromatography-mass spectrometry.In vitro, CCD-841CoN cell was placed in the ischemic solution under hypoxic conditions (94%N2,5%CO2,and 1%O2) in a 37 °C incubator to establish an ischemia and hypoxia model.The CCD-841CoN cells were divided into 7 groups, namely blank group and model group with normal rat serum plus control siRNA, tolvaptan group with rat serum containing tolvaptan plus control siRNA, KTBA group with rat serum containing KTBA plus control siRNA, KTBA plus p38MAPK siRNA group, KTBA plus p65NF-κB siRNA group,and KTBA plus AQP4siRNA group.After 24h and 48h of intervention with KTBA decoction,RT-qPCR,immunofluorescence and Western blot was used to detect the mRNA expression and protein expression levels of p38MAPK,I-κBα,p65NF-κB,AQP4, Occludin and ZO-1 in CCD-841CoN cells. RESULTS: Compared with the model, KTBA decoction improved the general state, decraesed the serum NT-proBNP level,HW/BW ratio, LVIDd and LVIDs, increased E-cadherin level,EF and FS,reduced number of collagen fibers deposited in the myocardial interstitium,and recovered irregular arrangement of myofibril and swollen or vacuolated mitochondria with broken crista in myocardium.Moreover, KTBA decoction inhibited the expression of p38MAPK,I-κBα,and p65NF-κB and upregulated AQP4, Occludin and ZO-1 in colon tissues and CCD-841CoN cells.Additionally,p38siRNA or SB203580, p65siRNA or PDTC, and AQP4siRNA partially weakened the protective effects of KTBA in vitro and vivo.Notably,The LEfSe analysis results showed that there were six gut biomaker bacteria in model group, including Allobaculum, Bacillales,Turicibacter, Turicibacterales,Turicibacteraceae,and Bacilli. Besides, three gut biomaker bacteria containing Deltaproteobacteria, Desulfovibrionaceae,and Desulfovibrionales were enriched by KTBA treatment in chronic HF model.There were five differential metabolites, including L-Leucine,Pelargonic acid, Capsidiol,beta-Carotene,and L- Erythrulose, which can be regulated back in the same changed metabolic routes by the intervention of KTBA.L-Leucine had the positive correlation with Bacillales, Turicibacterales,Turicibacteraceae,and Turicibacter.L-Leucine significantly impacts Protein digestion and absorption, Mineral absorption,and Central carbon metabolism in cancer regulated by KTBA, which is involved in the expression of MAPK and tight junction in intestinal epithelial cells. CONCLUSIONS: KTBA decoction manipulates the expression of several key proteins in the p38MAPK/p65NF-κB/AQP4 signaling pathway, modulates gut microbiota and metabolites toward a more favorable profile, improves gut barrier function, delays cardiomyocyte hypertrophy and fibrosis,and improves cardiac function.

Comparison of the components of fresh Panax notoginseng processed by different methods and their anti-anemia effects on cyclophosphamide-treated mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese herb Panax notoginseng (PN) tonifies blood, and its main active ingredient is saponin. PN is processed by different methods, resulting in different compositions and effects. AIM OF THE STUDY: To investigate changes in the microstructure and composition of fresh PN processed by different techniques and the anti-anemia effects on tumor-bearing BALB/c mice after chemotherapy with cyclophosphamide (CTX). MATERIALS AND METHODS: Fresh PN was processed by hot-air drying (raw PN, RPN), steamed at 120 °C for 5 h (steamed PN, SPN), or fried at 130 °C, 160 °C, or 200 °C for 8 min (fried PN, FPN1, FPN2, or FPN3, respectively); then, the microstructures were compared with 3D optical microscopy, quasi-targeted metabolites were detected by liquid chromatography tandem mass spectrometry (LC‒MS/MS), and saponins were detected by high-performance liquid chromatography (HPLC). An anemic mouse model was established by subcutaneous H22 cell injection and treatment with CTX. The antianemia effects of PN after processing via three methods were investigated by measuring peripheral blood parameters, performing HE staining and measuring cell proliferation via immunofluorescence. RESULTS: 3D optical profiling revealed that the surface roughness of the SPN and FPN was greater than that of the other materials. Quasi-targeted metabolomics revealed that SPN and FPN had more differentially abundant metabolites whose abundance increased, while SPN had greater amounts of terpenoids and flavones. Analysis of the composition and content of the targeted saponins revealed that the contents of rare saponins (ginsenoside Rh1, 20(S)-Rg3, 20(R)-Rg3, Rh4, Rk3, Rg5) were greater in the SPN. In animal experiments, the RBC, WBC, HGB and HCT levels in peripheral blood were increased by SPN and FPN. HE staining and immunofluorescence showed that H-SPN and M-FPN promoted bone marrow and spleen cell proliferation. CONCLUSION: The microstructure and components of fresh PN differed after processing via different methods. SPN and FPN ameliorated CTX-induced anemia in mice, but the effects of PN processed by these two methods did not differ.

Xinyang tablet alleviated cardiac dysfunction in a cardiac pressure overload model by regulating the receptor-interacting serum/three-protein kinase 3/FUN14 domain containing 1-mediated mitochondrial unfolded protein response and mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinyang tablet (XYT) has been used for heart failure (HF) for over twenty years in clinical practice, but the underlying molecular mechanism remains poorly understood. AIMS OF THE STUDY: In the present study, we aimed to explore the protective effects of XYT in HF in vivo and in vitro. MATERIALS AND METHODS: Transverse aortic constriction was performed in vivo to establish a mouse model of cardiac pressure overload. Echocardiography, tissue staining, and real-time quantitative PCR (qPCR) were examined to evaluate the protective effects of XYT on cardiac function and structure. Adenosine 5'-triphosphate production, reactive oxygen species staining, and measurement of malondialdehyde and superoxide dismutase was used to detect mitochondrial damage. Mitochondrial ultrastructure was observed by transmission electron microscope. Immunofluorescence staining, qPCR, and Western blotting were performed to evaluate the effect of XYT on the mitochondrial unfolded protein response and mitophagy, and to identify its potential pharmacological mechanism. In vitro, HL-1 cells and neonatal mouse cardiomyocytes were stimulated with Angiotensin II to establish the cell model. Western blotting, qPCR, immunofluorescence staining, and flow cytometry were utilized to determine the effects of XYT on cardiomyocytes. HL-1 cells overexpressing receptor-interacting serum/three-protein kinase 3 (RIPK3) were generated by transfection of RIPK3-overexpressing lentiviral vectors. Cells were then co-treated with XYT to determine the molecular mechanisms. RESULTS: In the present study, XYT was found to exerta protective effect on cardiac function and structure in the pressure overload mice. And it was also found XYT reduced mitochondrial damage by enhancing mitochondrial unfolded protein response and restoring mitophagy. Further studies showed that XYT achieved its cardioprotective role through regulating the RIPK3/FUN14 domain containing 1 (FUNDC1) signaling. Moreover, the overexpression of RIPK3 successfully reversed the XYT-induced protective effects and significantly attenuated the positive effects on the mitochondrial unfolded protein response and mitophagy. CONCLUSIONS: Our findings indicated that XYT prevented pressure overload-induced HF through regulating the RIPK3/FUNDC1-mediated mitochondrial unfolded protein response and mitophagy. The information gained from this study provides a potential strategy for attenuating mitochondrial damage in the context of pressure overload-induced heart failure using XYT.

Study on analgesic effect of Shentong Zhuyu Decoction in neuropathic pain rats by network pharmacology and RNA-Seq.

ETHNOPHARMACOLOGICAL RELEVANCE: Shentong Zhuyu Decoction (STZYD) is a traditional prescription for promoting the flow of Qi and Blood which is often used in the treatment of low back and leg pain clinicall with unclear mechanism. Neuropathic pain (NP) is caused by disease or injury affecting the somatosensory system. LncRNAs may play a key role in NP by regulating the expression of pain-related genes through binding mRNAs or miRNAs sponge mechanisms. AIM OF THE STUDY: To investigate the effect and potential mechanism of STZYD on neuropathic pain. METHODS: Chronic constriction injury (CCI) rats, a commonly used animal model, were used in this study. The target of STZYD in NP was analyzed by network pharmacology, and the analgesic effect of STZYD in different doses (H-STZYD, M-STZYD, L-STZYD) on CCI rats was evaluated by Mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL). Meanwhile, RNA-seq assay was used to detect the changed mRNAs and lncRNAs in CCI rats after STZYD intervention. GO analysis, KEGG pathway analysis, and IPA analysis were used to find key target genes and pathways, verified by qPCR and Western Blot. The regulatory effect of lncRNAs on target genes was predicted by co-expression analysis and ceRNA network construction. RESULTS: We found that STZYD can improve hyperalgesia in CCI rats, and H-STZYD has the best analgesic effect. The results of network pharmacological analysis showed that STZYD could play an analgesic role in CCI rats through the MAPK/ERK/c-FOS pathway. By mRNA-seq and lncRNA-seq, we found that STZYD could regulate the expression of Cnr1, Cacng5, Gucy1a3, Kitlg, Npy2r, and Grm8, and inhibited the phosphorylation level of ERK in the spinal cord of CCI rats. A total of 27 lncRNAs were associated with the target genes and 30 lncRNAs, 83 miRNAs and 5 mRNAs participated in the ceRNA network. CONCLUSION: STZYD has the effect of improving hyperalgesia in CCI rats through the MAPK/ERK/c-FOS pathway, which is related to the regulation of lncRNAs to Cnr1 and other key targets.

Mijiao formula regulates NAT10-mediated Runx2 mRNA ac4C modification to promote bone marrow mesenchymal stem cell osteogenic differentiation and improve osteoporosis in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The Mijiao (MJ) formula, a traditional herbal remedy, incorporates antlers as its primary constituent. It can effectively treat osteoporosis (OP), anti-aging, enhance immune activity, and change depression-like behavior. In this study, we investigated that MJ formula is a comprehensive treatment strategy, and may provide a potential approach for the clinical treatment of postmenopausal osteoporosis. AIM OF THE STUDY: The purpose of this study was to determine whether MJ formula promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and improved osteoporosis in ovariectomized rats by regulating the NAT10-mediated Runx2 mRNA ac4C modification. MATERIALS AND METHODS: Female Sprague-Dawley (SD) rats were used to investigate the potential therapeutic effect of MJ formula on OP by creating an ovariectomized (OVX) rat model. The expression of osteogenic differentiation related proteins in BMSCs was detected in vivo, indicating their role in promoting bone formation. In addition, the potential mechanism of its bone protective effect was explored via in vitro experiments. RESULTS: Our study showed that MJ formula significantly mitigated bone mass loss in the OVX rat model, highlighting its potential as an OP therapeutic agent. We found that the possible mechanism of action was the ability of this formulation to stabilize Runx2 mRNA through NAT10-mediated ac4C acetylation, which promoted osteogenic differentiation of BMSCs and contributed to the enhancement of bone formation. CONCLUSIONS: MJ formula can treat estrogen deficiency OP by stabilizing Runx2 mRNA, promoting osteogenic differentiation and protecting bone mass. Conceivably, MJ formulation could be a safe and promising strategy for the treatment of osteoporosis.

Jiawei Dachaihu decoction protects against mitochondrial dysfunction in atherosclerosis (AS) mice with chronic unpredictable mild stress (CUMS) via SIRT1/PGC-1α/TFAM/LON signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: JiaWei DaChaiHu is composed of Bupleurum chinense, Scutellaria baicalensis, Pinellia ternata, Paeonia lactiflora, Zingiber officinaleRoscoe, Poncirus tuifoliata, Rheum palmatum L., Curcumae Radix, Herba Lysimachiae, Ziziphus. JiaWei DaChaiHu is one of the most common traditional Chinese medicines for the treatment of depression. AIM OF THE STUDY: The chronic unpredictable mild stress (CUMS) has been shown to promote atherosclerosis (AS). Dachaihu has been widely used in traditional Chinese medicine and has been known to exert distinct pharmacological effects. This investigation aims to examine the therapeutic effect of Jiawei Dachaihu extract on AS animal models with CUMS. METHODS: AS-CUMS mice model was established by Apoe-/- mice. Mice were treated with Jiawei Dachaihu. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C) levels were measured using ELISA kits. Aortic tissue pathologic changes detected by oil red O staining. Mice behavioral changes detected by sucrose preference test and sucrose preference test. The relative mRNA expression levels of CRH, ND1, and TFAM were determined by qRT-PCR. 5-HT1A, BDNF, LON, TFAM, PGC-1α, and SIRT1 protein expression determined by western blotting. ATP content detected by ATP kits. RESULTS: The treatment with Jiawei Dachaihu extract alleviated the veins plaque and reduced stress signs in vitro and in vivo. It increased the ATP and HDL-C levels while decreased the TC, TG, LDL-C levels. Jiawei Dachaihu extract treatment upregulated Lon, SIRT1, TFAM, PGC-1α, BDNF, and 5-HT1A protein expression and regained mitochondrial function. CONCLUSION: Jiawei Dachaihu extract could alleviate AS and reduce CUMS by upregulating the SIRT1/PGC-1α signaling and promoted its crosstalk with Lon protein to maintain mitochondrial stability.

Rosmarinic acid alleviates toosendanin-induced liver injury through restoration of autophagic flux and lysosomal function by activating JAK2/STAT3/CTSC pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a natural polyphenol abundant in numerous herbal remedies, has been attracting growing interest owing to its exceptional ability to protect the liver. Toosendanin (TSN), a prominent bioactive compound derived from Melia toosendan Siebold & Zucc., boasts diverse pharmacological properties. Nevertheless, TSN possesses remarkable hepatotoxicity. Intriguingly, the potential of RA to counteract TSN-induced liver damage and its probable mechanisms remain unexplored. AIM OF THE STUDY: This study is aimed at exploring whether RA can alleviate TSN-induced liver injury and the potential mechanisms involved autophagy. MATERIALS AND METHODS: CCK-8 and LDH leakage rate assay were used to evaluate cytotoxicity. Balb/c mice were intraperitoneally administered TSN (20 mg/kg) for 24 h after pretreatment with RA (0, 40, 80 mg/kg) by gavage for 5 days. The autophagic proteins P62 and LC3B expressions were detected using western blot and immunohistochemistry. RFP-GFP-LC3B and transmission electron microscopy were applied to observe the accumulation levels of autophagosomes and autolysosomes. LysoTracker Red and DQ-BSA staining were used to evaluate the lysosomal acidity and degradation ability respectively. Western blot, immunohistochemistry and immunofluorescence staining were employed to measure the expressions of JAK2/STAT3/CTSC pathway proteins. Dual-luciferase reporter gene was used to measure the transcriptional activity of CTSC and RT-PCR was used to detect its mRNA level. H&E staining and serum biochemical assay were employed to determine the degree of damage to the liver. RESULTS: TSN-induced damage to hepatocytes and livers was significantly alleviated by RA. RA markedly diminished the autophagic flux blockade and lysosomal dysfunction caused by TSN. Mechanically, RA alleviated TSN-induced down-regulation of CTSC by activating JAK2/STAT3 signaling pathway. CONCLUSION: RA could protect against TSN-induced liver injury by activating the JAK2/STAT3/CTSC pathway-mediated autophagy and lysosomal function.

Xin-Yi-Qing-Fei-Tang and its critical components reduce asthma symptoms by suppressing GM-CSF and COX-2 expression in RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) XYQFT is composed of 10 herbs. According to the NHIRD, XYQFT is one of the top ten most commonly used TCM prescriptions for asthma treatment. AIM OF THE STUDY: The aim of this study was to explore whether XYQFT reduces asthma symptoms in a mouse model of chronic asthma and determine the immunomodulatory mechanism of mast cells. MATERIALS AND METHODS: BALB/c mice were intratracheally (it) stimulated with 40 µL (2.5 µg/µL) of Dermatophagoides pteronyssinus (Der p) once a week for 6 consecutive weeks and orally administered XYQFT at 1 g/kg 30 min before Der p stimulation. Airway hypersensitivity, inflammatory cells in the BALF and total IgE in the blood were assessed in mice. In addition, RBL-2H3 cells (mast cells) were stimulated with DNP-IgE, after which different concentrations of XYQFT were added for 30 min to evaluate the effect of XYQFT on the gene expression and degranulation of DNP-stimulated RBL-2H3 cells. After the compounds in XYQFT were identified using LC‒MS/MS, the PBD method was used to identify the chemical components that inhibited the expression of the GM-CSF and COX-2 genes in mast cells. RESULTS: The airway hypersensitivity assay demonstrated that XYQFT significantly alleviated Der p-induced airway hypersensitivity. Moreover, cell counting and typing of bronchoalveolar lavage fluid revealed a significant reduction in Der p-induced inflammatory cell infiltration with XYQFT treatment. ELISA examination further indicated a significant decrease in Der p-induced total IgE levels in serum following XYQFT administration. In addition, XYQFT inhibited the degranulation and expression of genes (IL-3, IL-4, ALOX-5, IL-13, GM-CSF, COX-2, TNF-α, and MCP-1) in RBL-2H3 cells after DNP stimulation. The compounds timosaponin AIII and genkwanin in XYQFT were found to be key factors in the inhibition of COX-2 and GM-CSF gene expression in mast cells. CONCLUSION: By regulating mast cells, XYQFT inhibited inflammatory cell infiltration, airway hypersensitivity and specific immunity in a mouse model of asthma. In addition, XYQFT synergistically inhibited the expression of the GM-CSF and COX-2 genes in mast cells through timosaponin AIII and genkwanin.

Active fractions from Jingfang Baidu Powder alleviate Klebsiella-induced Pneumonia by inhibiting TLR4/Myd88-ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jingfang Baidu Powder (JFBDP) is a classic traditional Chinese medicine prescription. Although Jingfang Baidu powder obtained a general consensus on clinical efficacy in treating pneumonia, there were many Chinese herbal drugs in formula, complex components, and large oral dosage, which brings certain obstacles to clinical application. AIM OF THE STUDY: Therefore, screening of the active fraction that exerts anti-pneumonia helps improve the pharmaceutical preparation, improve the treatment compliance of patients, and further contribute to the clinical application, and the screening of the new active ingredients with anti-pneumonia. The histopathological observation, real-time quantitative PCR, western blotting, and immunofluorescence were applied to evaluate the anti-pneumonia efficacy of active fractions from JFBDP. RESULTS: Three fractions from JFBDP inhibit the gene expression of IL-1ß, IL-10, CCL3, CCL5, and CCL22 in lung tissue infected by Klebsiella at various degrees, and presented a good dose-response relationship. JF50 showed stronger anti-inflammatory effects among three fractions including JF30, JF50, and JF75. Besides, JF50 significantly reduced the protein expression of TLR4 and Myd88 in lung tissue infected with Klebsiella, and it also significantly inhibited p-ERK and p-NF-κB p65. JF50 significantly inhibits the protein expression of Caspase 3, Caspase 8, and Caspase 9 in lung tissue infected with Klebsiella at the dose of 25 mg/kg and 50 mg/kg. CONCLUSION: JF50 improves lung pathological damage in Klebsiella pneumonia mice by inhibiting the TLR4/Myd88/NF-κB-ERK signaling pathway, and inhibiting apoptosis of lung tissue cells. These findings provide a reference for further exploring the active substance basis of Jingfang Baidu Powder in treating bacterial pneumonia.

Mechanism and functional substances of Saiga antelope horn in treating hypertension with liver-yang hyperactivity syndrome explored using network pharmacology and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Saiga antelope horn (SAH) is a traditional Chinese medicine for treating hypertension with liver-yang hyperactivity syndrome (Gan-Yang-Shang-Kang, GYSK), that has a long history of clinical application and precise efficacy, but its mechanism and functional substances are still unknown. Based on the demand for alternative research on the rare and endangered SAH, the group designed and carried out the following studies. AIM OF THE STUDY: The purpose of this research was to demonstrate the functional substances and mechanisms of SAH in the treatment of GYSK hypertension. MATERIALS AND METHODS: The GYSK-SHR model was constructed by administering a decoction of aconite to spontaneously hypertensive rats (SHRs). Blood pressure (BP), behavioural tests related to GYSK, and pathological changes in the kidneys, heart and aorta were measured to investigate the effects of SAH on GYSK-SHRs. Proteomic analysis was used to identify the keratins and peptides of SAH. Moreover, network pharmacology and plasma metabolomics studies were carried out to reveal the mechanisms by which functional peptides in SAH regulate GYSK-hypertension. RESULTS: SAH has a significant antihypertensive effect on GYSK hypertensive animals. It has also been proven to be effective in protecting the function and structural integrity of the kidneys, heart and aorta. Moreover, SAH improved the abnormalities of 31 plasma biomarkers in rats. By constructing a "biomarker-target-peptide" network, 10 functional peptides and two key targets were screened for antihypertensive effects of SAH. The results indicated that SAH may exert a therapeutic effect by re-establishing the imbalance of renin-angiotensin (RAS) system. CONCLUSIONS: Functional peptides from keratin contained in SAH are the main material basis for the treatment of GYSK-hypertension and exhibited the protective effect on the GYSK-SHR model through the RAS system.

Qilong capsule prevents myocardial ischemia/reperfusion injury by inhibiting platelet activation via the platelet CD36 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qilong capsule (QC) is developed from the traditional Chinese medicine formula Buyang Huanwu Decoction, which has been clinically used to invigorate Qi and promote blood circulation to eliminate blood stasis. Myocardial ischemia‒reperfusion injury (MIRI) can be attributed to Qi deficiency and blood stasis. However, the effects of QC on MIRI remain unclear. AIM OF THE STUDY: This study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. MATERIALS AND METHODS: The left anterior descending artery of adult Sprague‒Dawley rats was ligated for 30 min and then reperfused for 120 min with or without QC treatment. Then, the whole blood viscosity, plasma viscosity, coagulation, platelet adhesion rate, platelet aggregation, and platelet release factors were evaluated. Platelet CD36 and its downstream signaling pathway-related proteins were detected by western blotting. Furthermore, the active components of QC and the molecular mechanism by which QC regulates platelet function were assessed via molecular docking, platelet aggregation tests in vitro and BLI analysis. RESULTS: We found that QC significantly reduced the whole blood viscosity, plasma viscosity, platelet adhesion rate, and platelet aggregation induced by ADP or AA in rats with MIRI. The inhibition of platelet activation by QC was associated with reduced levels of ß-TG, PF-4, P-selectin and PAF. Mechanistically, QC effectively attenuated the expression of platelet CD36 and thus inhibited the activation of Src, ERK5, and p38. The active components of QC apparently suppressed platelet aggregation in vitro and regulated the CD36 signaling pathway. CONCLUSIONS: QC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

Zhilong Huoxue Tongyu Capsule regulates the macrophage polarization and inflammatory response via the let-7i/TLR9/MyD88 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS. AIM OF THE STUDY: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment. MATERIALS AND METHODS: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay. RESULTS: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor ß1 (TGF-ß1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i. CONCLUSIONS: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS.

Multi-omics and chemical profiling approaches to understand the material foundation and pharmacological mechanism of sophorae tonkinensis radix et rhizome-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophorae tonkinensis Radix et Rhizoma (STR) is an extensively applied traditional Chinese medicine (TCM) in southwest China. However, its clinical application is relatively limited due to its hepatotoxicity effects. AIM OF THE STUDY: To understand the material foundation and liver injury mechanism of STR. MATERIALS AND METHODS: Chemical compositions in STR and its prototypes in mice were profiled by ultra-performance liquid chromatography coupled quadrupole-time of flight mass spectrometry (UPLC-Q/TOF MS). STR-induced liver injury (SILI) was comprehensively evaluated by STR-treated mice mode. The histopathologic and biochemical analyses were performed to evaluate liver injury levels. Subsequently, network pharmacology and multi-omics were used to analyze the potential mechanism of SILI in vivo. And the target genes were further verified by Western blot. RESULTS: A total of 152 compounds were identified or tentatively characterized in STR, including 29 alkaloids, 21 organic acids, 75 flavonoids, 1 quinone, and 26 other types. Among them, 19 components were presented in STR-medicated serum. The histopathologic and biochemical analysis revealed that hepatic injury occurred after 4 weeks of intragastric administration of STR. Network pharmacology analysis revealed that IL6, TNF, STAT3, etc. were the main core targets, and the bile secretion might play a key role in SILI. The metabolic pathways such as taurine and hypotaurine metabolism, purine metabolism, and vitamin B6 metabolism were identified in the STR exposed groups. Among them, taurine, hypotaurine, hypoxanthine, pyridoxal, and 4-pyridoxate were selected based on their high impact value and potential biological function in the process of liver injury post STR treatment. CONCLUSIONS: The mechanism and material foundation of SILI were revealed and profiled by a multi-omics strategy combined with network pharmacology and chemical profiling. Meanwhile, new insights were taken into understand the pathological mechanism of SILI.

Attenuating Renal Interstitial Fibrosis by Shenqi Pill via Reducing Inflammation Response Regulated by NF-κB Pathway In Vitro and In Vivo.

INTRODUCTION: One of the most significant clinical features of chronic  kidney disease is renal interstitial fibrosis (RIF). This study aimed  to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. METHODS: RIF model was established by conducting unilateral  ureteral obstruction (UUO) surgery on rat or stimulating human  kidney-2 (HK-2) cell with transforming growth factor ß1 (TGFß1).  After modeling, the rats in the SQP low dose group (SQP-L), SQP  middle dose group (SQP-M) and SQP high dose group (SQP-H)  were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the  TGFß1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing  serum. In in vivo assays, serum creatinine (SCr) and blood urea  nitrogen (BUN) content were measured, kidney histopathology  was evaluated., and α-smooth muscle actin (α-SMA) expression  was detected by immunohistochemistry. Interleukin-1ß (IL-1ß),  interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content,  inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were  assessed. Meanwhile, cell viability, inflammatory cytokines content,  α-SMA expression, IKBα and P65 phosphorylation were detected  in vitro experiment.  Results. SQP exhibited reno-protective effect by decreasing SCr  and BUN content, improving renal interstitial damage, blunting  fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after  the treatment with SQP-containing serum, viability and α-SMA  expression were remarkably decreased in TGFß1-stimulated HK-2  cell. Furthermore, SQP markedly down-regulated IL-1ß, IL-6, and  TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro.  Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa  B (NF-κB) pathway related inflammatory response, which indicates  that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

Investigation of the underlying mechanism of Buyang Huanwu decoction in ischemic stroke by integrating systems pharmacology-proteomics and in vivo experiments.

Buyang Huanwu Decoction (BHD) has been effective in treating ischemic stroke (IS). However, its mechanism of action remains unclear. The study intended to explore the potential mechanism of BHD against IS using systems pharmacology, proteomics, and animal experiments. The active components of BHD were identified from UPLC-Q-TOF-MS and literature mining. Systems pharmacology and proteomics were employed to investigate the underlying mechanism of BHD against IS. The AutoDock tool was used for molecular docking. A middle cerebral artery occlusion (MCAO) model rat was utilized to explore the therapeutic benefits of BHD. The rats were divided into sham, model, BHD (5, 10, 20 g/kg, ig) groups. The neurological scores, pathological section characteristics, brain infarct volumes, inflammatory cytokines, and signaling pathways were investigated in vivo experiments. The results of systems pharmacology showed that 13 active compounds and 112 common targets were screened in BHD. The docking results suggested that the active compounds in BHD had a high affinity for the key targets. In vivo experiments demonstrated that BHD exhibited neuroprotective benefits by lowering the neurological score, the volume of the cerebral infarct, the release of inflammatory cytokines, and reducing neuroinflammatory damage in MCAO rats. Furthermore, BHD decreased TNF-α and CD38 levels while increasing ATP2B2, PDE1A, CaMK4, p-PI3K, and p-AKT. Combined with systems pharmacology and proteomic studies, we confirmed that PI3K-Akt and calcium signaling pathways are the key mechanisms for BHD against IS. Furthermore, this study demonstrated the feasibility of combining proteomics with systems pharmacology to study the mechanism of herbal medicine.

Lycium Barbarum Polysaccharides Improves Cognitive Functions in ICV-STZ-Induced Alzheimer's Disease Mice Model by Improving the Synaptic Structural Plasticity and Regulating IRS1/PI3K/AKT Signaling Pathway.

Lycium barbarum polysaccharide (LBP) have a certain curative effect on hypoglycemic and neuroprotective effects, but the specific mechanism is unclear and needs to be further explored. This study aimed to clarify the mechanisms of LBP in the treatment of ICV-STZ mice model of AD from the perspectives of insulin resistance, IRS1/PI3K/AKT signaling pathway, and synaptic protein expression. We used male C57BL/6J mice injected with STZ (3 mg/kg) in the lateral ventricle as an AD model. After treatment with LBP, the learning and memory abilities of ICV-STZ mice were enhanced, and the pathological changes in brain tissue were alleviated. LBP can regulate the expression of proteins related to the IRS1/PI3K/AKT signaling pathway and thereby reducing Aß deposition and tau protein phosphorylation in the brain of ICV-STZ mice. In addition, LBP also can up-regulate the expression of synaptic proteins. The results indicated that LBP played a neuroprotective role by regulating the IRS1/PI3K/AKT pathway, inhibiting tau protein hyperphosphorylation and improving the expression levels of synapse-related proteins.