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.

Systematic identification of the interventional mechanism of Qingfei Xiaoyan Wan (QFXYW) in treatment of the cytokine storm in acute lung injury using transcriptomics-based system pharmacological analyses.

CONTEXT: Acute lung injury (ALI) is a complex, severe inflammation disease with high mortality, and there is no specific and effective treatment for ALI. Qingfei Xiaoyan Wan (QFXYW) has been widely used to treat lung-related diseases for centuries. OBJECTIVE: This study evaluates the potential effects and elucidates the therapeutic mechanism of QFXYW against LPS induced ALI in mice. MATERIALS AND METHODS: BALB/c Mice in each group were first orally administered medicines (0.9% saline solution for the control group, 0.5 mg/kg Dexamethasone, or 1.3, 2.6, 5.2 g/kg QFXYW), after 4 h, the groups were injected LPS (1.0 mg/kg) to induce ALI, then the same medicines were administered repeatedly. The transcriptomics-based system pharmacological analyses were applied to screen the hub genes, RT-PCR, ELISA, and protein array assay was applied to verify the predicted hub genes and key pathways. RESULTS: QFXYW significantly decreased the number of leukocytes from (6.34 ± 0.51) × 105/mL to (4.01 ± 0.11) × 105/mL, accompanied by the neutrophil from (1.41 ± 0.19) × 105/mL to (0.77 ± 0.10) × 105/mL in bronchoalveolar lavage fluid (BALF). Based on Degree of node connection (Degree) and BottleNeck (BN), important parameters of network topology, the protein-protein interaction (PPI) network screened hub genes, including IL-6, TNF-α, CCL2, TLR2, CXCL1, and MMP-9. The results of RT-PCR, ELISA, and protein chip assay revealed that QFXYW could effectively inhibit ALI via multiple key targets and the cytokine-cytokine signalling pathway. CONCLUSIONS: This study showed that QFXYW decreased the number of leukocytes and neutrophils by attenuating inflammatory response, which provides an important basis for the use of QFXYW in the treatment of ALI.

Integration of transcriptomics and system pharmacology to reveal the therapeutic mechanism underlying Qingfei Xiaoyan Wan to treat allergic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Asthma is a chronic inflammatory disease, characterized by airway inflammation, hyperresponsiveness, and bronchial smooth muscle contraction. Qingfei Xiaoyan Wan (QFXYW), a traditional Chinese formula, has been shown to exert anti-asthma effects and immune response in multiple diseases. AIM OF THIS STUDY: In this study, we evaluated the therapeutic mechanism of QFXYW in the suppression of allergic asthma by integrating of transcriptomics and system pharmacology. MATERIALS AND METHODS: BALB/c mice were sensitized with ovalbumin (OVA) to establish the allergic asthma model, and its success was confirmed with behavioral observations. Lung histopathological analysis, inflammatory pathology scores, transcription factors were used to evaluate the effects of QFXYW on allergic asthma. The therapeutic mechanism of QFXYW in treating allergic asthma through integrated transcriptomics and system pharmacology was then determined: hub genes were screened out by topological analysis and functional enrichment analysis were performed to identify key signaling pathway. Subsequently, quantitative RP-PCR and protein array were performed to detect the mRNA of hub genes and to predict the key pathway in OVA-induced allergic asthma, respectively. RESULTS: Our results demonstrated that QFXYW could significantly attenuate inflammatory cell infiltration, mucus secretion, and epithelial damage. The transcriptomics analysis found the six hub genes with the highest values- CXCL10, CXCL2, CXCL1, IL-6, CCL-5, and CCL-4 were screened out. Functional enrichment analysis showed that the differentially expressed genes (DEGs) were mainly enriched in the inflammatory response and cytokine signaling pathway. Moreover, the quantitative RT-PCR verification experiment found the CXCL2 and CXCL1 were significantly suppressed after treatment with QFXYW. The results of protein array showed that QFXYW inhibited the multi-cytokines of OVA-induced allergic asthma via cytokine signaling pathway. CONCLUSIONS: QFXYW may have mediated OVA-induced allergic asthma mainly through the hub genes CXCL2, CXCL1, and the cytokine signaling pathway. This finding will offer a novel strategy to explore effective and safe mechanism of Traditional Chinese Medicine (TCM) formula to treat allergic asthma.

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

[Rapid PCR authentication Lonicera japanica].

To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.