[Anti-aging effect and molecular mechanism of Xiyangshen Sanqi Danshen Granules based on metabolomics and bioinformatics].

This study investigated the anti-aging mechanism of Xiyangshen Sanqi Danshen Granules based on metabonomics, network pharmacology, and molecular docking. The aging mice model was induced by intraperitoneal injection of D-galactose(D-gal). Mice were randomly divided into a control group, model group, melatonin group(MT group), and low, medium, and high dose groups of Xiyangshen Sanqi Danshen Granules(XSD-L, XSD-M, and XSD-H). An open-field experiment was conducted, and the expression of cell cycle arrest proteins(p16) and phosphorylated histone family 2A variant(γH2AX) in the brain tissue was detected by immunofluorescence. The expression of interleukin-1ß(IL-1ß) and interleukin-6(IL-6) in the brain tissue was detected by enzyme-linked immunosorbent assay(ELISA). Metabolomics analysis was performed on the serum of mice in control, model, and XSD-H groups to obtain metabolic processes and metabolites. The effective chemical components and potential targets of Xiyangshen Sanqi Danshen Granules were predicted through network pharmacology, and the network diagram of "drug-effective chemical components-key targets" was constructed. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis were carried out, and a protein-protein interaction(PPI) network was constructed to clarify the anti-aging mechanism of Xiyangshen Sanqi Danshen Granules. The results showed that the Xiyangshen Sanqi Danshen Granules could significantly improve the aging degree of D-gal mice, significantly improve the total motion distance and the mean motion speed of D-gal mice, and reduce the rest time. In addition, Xiyangshen Sanqi Danshen Granules could significantly reduce the protein levels of IL-6 and IL-1ß and the expression of p16 and γH2AX in D-gal mice. Compared with the model group, 66 differential metabolites(DMs) were significantly up-regulated, and 91 DMs were down-regulated in the XSD-H group. Moreover, four key metabolic pathways(tryptophan metabolism, glycerophospholipid metabolism, pyrimidine metabolism, and lysine degradation) and 16 biomarkers(lysine, tryptophan, indoleacetaldehyde, PCs, LysoPCs, 3-hydroxyanthranilic acid, melatonin, etc) were screened out. 58 main active components and 62 key targets of Xiyangshen Sanqi Danshen Granules were screened by network pharmacology. The GO functional enrichment analysis found the positive regulation of gene expression, drug response, etc. KEGG pathway enrichment screening involved diabetic complications-related AGE-RAGE signaling pathway, hypoxia inducible factor-1 signaling pathway, etc. Through the PPI network and molecular docking, six potential core targets of STAT3, MAPK1, MAPK14, EGFR, FOS, and STAT1 were screened.

Single-cell RNA sequencing reveals that VIM and IFITM3 are vital targets of Dengzhan Shengmai capsule to protect against cerebral ischemic injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is one of the leading causes of mortality, but therapies are limited. Dengzhan Shengmai capsule (DZSM) was included by the Chinese Pharmacopoeia 2020 and has been broadly used for the treatment of ischemic stroke. However, the mechanism of DZSM against ischemic stroke is unclear. AIM OF THE STUDY: This study used RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to investigate the mechanism of action of DZSM against ischemic stroke. MATERIALS AND METHODS: The rats were randomly divided into six groups: the Sham, I/R (water), I/R + DZSM-L (0.1134g/kg), I/R + DZSM-H (0.4536g/kg), I/R + NMDP (20mg/kg), and I/R + Ginaton (20mg/kg). The rats were administrated drugs for 5 days then followed by the ischemic brain injury caused by middle cerebral artery occlusion (MCAO). The neuroprotective effect was assessed by infraction rate, neurological deficit scores, regional cerebral blood flow (rCBF), hematoxylin and eosin (H&E) staining, and Nissl staining. Based on RNA-seq and scRNA-seq, the vital biological processes and core targets of DZSM against cerebral ischemia were revealed. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) staining were used to investigate the vital biological processes and core targets of DZSM against ischemic stroke. RESULTS: Administration of DZSM significantly reduced the infarction rate and Zea Longa score, Garcia JH score, and ameliorated the reduction in rCBF. And alleviated the neuronal damage, such as increased neuronal density level and Nissl bodies density level. RNA-seq analysis revealed that DZSM played important roles in inflammation and apoptosis. ELISA and IF straining validation confirmed that DZSM significantly decreased the expression of IL-6, IL-1ß, TNF-α, ICAM-1, IBA-1, MMP9, and Cleaved caspase-3 in MCAO rats. ScRNA-seq analysis identified 8 core targets in neurons including HSPB1, SPP1, MT2A, GFAP, IFITM3, VIM, CRIP1, and GPD1, and VIM and IFITM3 was verified to be decreased by DZSM in neurons. CONCLUSION: Our study illustrates the neuroprotective effect of DZSM against ischemia stroke, and VIM and IFITM3 were identified as vital targets in neurons of DZSM in protecting against MCAO-induced I/R injury.

Multi-omics reveals Dengzhan Shengmai formulation ameliorates cognitive impairments in D-galactose-induced aging mouse model by regulating CXCL12/CXCR4 and gut microbiota.

Dengzhan Shengmai (DZSM), a traditional Chinese medicine formulation, has been administered extensively to elderly individuals with cognitive impairment (CI). However, the underlying mechanisms by which Dengzhan Shengmai improves cognitive impairment remains unknown. This study aimed to elucidate the underlying mechanism of the effect of Dengzhan Shengmai on aging-associated cognitive impairment via a comprehensive combination of transcriptomics and microbiota assessment. Dengzhan Shengmai was orally administered to a D-galactose-induced aging mouse model, and evaluation with an open field task (OFT), Morris water maze (MWM), and histopathological staining was performed. Transcriptomics and 16S rDNA sequencing were applied to elucidate the mechanism of Dengzhan Shengmai in alleviating cognitive deficits, and enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (PCR), and immunofluorescence were employed to verify the results. The results first confirmed the therapeutic effects of Dengzhan Shengmai against cognitive defects; specifically, Dengzhan Shengmai improved learning and impairment, suppressed neuro loss, and increased Nissl body morphology repair. Comprehensive integrated transcriptomics and microbiota analysis indicated that chemokine CXC motif receptor 4 (CXCR4) and its ligand CXC chemokine ligand 12 (CXCL12) were targets for improving cognitive impairments with Dengzhan Shengmai and also indirectly suppressed the intestinal flora composition. Furthermore, in vivo results confirmed that Dengzhan Shengmai suppressed the expression of CXC motif receptor 4, CXC chemokine ligand 12, and inflammatory cytokines. This suggested that Dengzhan Shengmai inhibited CXC chemokine ligand 12/CXC motif receptor 4 expression and modulated intestinal microbiome composition by influencing inflammatory factors. Thus, Dengzhan Shengmai improves aging-related cognitive impairment effects via decreased CXC chemokine ligand 12/CXC motif receptor 4 and inflammatory factor modulation to improve gut microbiota composition.

Integrating RNA-sequencing and network analysis to explore the mechanism of topical Pien Tze Huang treatment on diabetic wounds.

Introduction: Diabetic ulcers have become one of the major complications of diabetes mellitus (DM) and are a leading cause of death and disabling disease. However, current therapies are not effective enough to meet clinical needs. A traditional Chinese medicine (TCM) formula, Pien Tze Huang (PZH), is known as a medicine that is used to treat diabetic ulcers. Methods: In this study, PZH (0.05 g/cm2 and 0.15 g/cm2) and the positive drug-rhEGF were topically administered in a high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic full-thickness incisional wounds, respectively. Wound healing was assessed by wound closure rate, two-photon microscope (SHG), staining with Hematoxylin and eosin (H&E), and Masson's trichrome (MTC). Then, RNA sequencing (RNA-seq) analysis, Enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence (IF), network analysis, were performed. Results and discussion: The results showed that PZH significantly accelerated wound healing, as well as enhanced the expression of collagen. RNA-seq analysis showed that PZH has functions on various biological processes, one of the key biological processes is inflammatory response. Tlr9, Klrk1, Nod2, Tlr2, and Ifng were identified as vital targets and the NF-κB signaling pathway was identified as the vital pathway. Additionally, PZH profoundly reduced the levels of Cleaved caspase-3 and promoted the expression of CD31 and TGF-ß1. Mechanically, PZH significantly decreased expression of NKG2-D, NOD2, and TLR2, and further inhibited the activation of downstream NF-κB signaling pathway and inhibited expression of inflammatory factors (IFN-γ and IL-1ß). Importantly, we found that several active ingredients may play a significant role in diabetic wound healing, including Notoginsenoside R1, Deoxycorticosterone, Ursolic acid, and 4-Methoxyphenol. In summary, our study sheds light on the complicated mechanisms underlying the promising anti-diabetic wounds of PZH and provides the discovery of agents treating diabetic ulcers.

Huangbai liniment and berberine promoted wound healing in high-fat diet/Streptozotocin-induced diabetic rats.

Diabetic ulcer is a challenging complication of diabetes mellitus but current treatments cannot achieve satisfactory results. In this study, the effect of Huangbai liniment (HB) and berberine on the wound healing in high fat diet/streptozotocin injection induced diabetic rats was investigated by RNA-seq technology. HB topical treatment promoted wound healing in the diabetic patients and diabetic rats, and it affected multiple processes, of which IL-17 signalling pathway was of importance. Inhibiting IL-17a by its inhibitor or antibody remarkably facilitated wound healing and HB significantly repressed the high IL-17 expression and its downstream targets, including Cxcl1, Ccl2, Mmp3, Mmp9, G-CSF, IL1B and IL6, in diabetic wounds, promoted T-AOC, SOD activity and GSH levels; decreased the levels of nitrotyrosine and 8-OHdG; enhanced angiogenesis-related CD31, PDGF-BB and ANG1 expression; inhibited cleaved caspase-3 levels and promoted TIMP1 and TGFB1. Moreover, berberine (a major component in HB) repressed the IL-17 signalling pathway, and promoted wound healing in diabetes mellitus. This study highlights the strategy of targeting IL-17a in diabetic wounds, deepens the understanding of wound healing in diabetes mellitus in a dynamic way and reveals the characteristics of HB and berberine in promoting wound healing of type 2 diabetes mellitus.

Integrated transcriptomics and metabolomics analysis reveals that C3 and C5 are vital targets of DuZhi Wan in protecting against cerebral ischemic injury.

BACKGROUND/AIMS: Duzhi Wan (DZW) has been extensively used in the prevention and treatment of ischemic stroke, but the mechanisms underlying its effects remain unclear. In this study, a combination of transcriptomics, metabolomics and network analysis was applied to identify the preventive mechanism of DZW in middle cerebral artery occlusion (MCAO)-induced ischemia/reperfusion (I/R) injury. METHODS: The mice were divided into five groups: the sham group, I/R group, I/R + Ginaton group, I/R+DZW-L group, and I/R+DZW-H group. Neurological deficit scores and regional cerebral blood flow (rCBF), hematoxylin and eosin (H&E) and Nissl staining results were evaluated. Transcriptomics analysis and metabolomics analysis were applied to identify the key genes and metabolites, and qRT-PCR, ELISA, and immunofluorescence were applied to verify the key targets. RESULTS: DZW significantly decreased the infarction size and neurological deficit scores, increased the rCBF percentage and neuronal number and improved neuronal morphology after MCAO. Transcriptomics and metabolomics analysis revealed that C3 and C5ar1 were core targets of DZW and indirectly regulated downstream purine metabolism, the pentose phosphate pathway, and glycerophospholipid metabolism-associated pathways via inflammatory cells. Moreover, ELISA, qRT-PCR, and immunofluorescence further confirmed that DZW significantly decreased the expression of C3, C5ar1, C5 and downstream inflammatory cytokines, including IL-6, IL-1ß and MMP-9, at the gene and protein levels, suggesting that DZW decreased neuroinflammation and inhibited related metabolic pathways. CONCLUSION: C3 and C5 play important roles in the neuroprotective and antineuroinflammatory effects of DZW in protecting against cerebral I/R. This study provides novel insights into the neuroprotective effects of DZW and its clinical application.