Exploration of the mechanism of Taohong Siwu Decoction for the treatment of ischemic stroke based on CCL2/CCR2 axis.

Background and aims: Taohong Siwu Decoction (THSWD) is a traditional Chinese herbal prescription that is effective for ischemic stroke, Whether THSWD regulates the CCL2/CCR2 axis and thus reduces the inflammatory response induced by ischemic stroke is not known. The aim of this study was to elucidate the mechanism of action of THSWD in the treatment of ischemic stroke using bioinformatics combined with in vitro and in vivo experiments. Methods: R language was used to analyze middle cerebral artery occlusion/reperfusion (MCAO/R) rat transcriptome data and to identify differential gene expression following THSWD treatment. Gene set enrichment analysis (GSEA) was used to analyze the gene set enrichment pathway of MCAO/R rats treated with THSWD. PPI networks screened key targets. The Human Brain Microvascular Endothelial Cells (HBMEC) Oxygen Glucose Deprivation/Reoxygenation (OGD/R) model and SD rat models of MCAO/R were established. FITC-dextran, immunofluorescence, flow cytometry, ELISA, immunohistochemistry, Western blotting, and RT-qPCR were performed to identify potential treatment targets. Results: A total of 515 differentially expressed genes of THSWD in MCAO/R rats were screened and 92 differentially expressed genes of THSWD potentially involved in stroke intervention were identified, including Cd68, Ccl2, and other key genes. In vitro, THSWD reversed the increase in permeability of HBMEC cells and M1/M2 polarization of macrophages induced by CCL2/CCR2 axis agonists. In vivo, THSWD improved nerve function injury and blood-brain barrier injury in MCAO/R rats. Further, THSWD inhibited the infiltration and polarization of macrophages, reduced the expression of IL-6, TNF-α, and MMP-9, and increased the expression of IL-4, while reducing the gene and protein expression of CCL2 and CCR2. Conclusion: THSWD may play a protective role in ischemic stroke by inhibiting the CCL2/CCR2 axis, reducing the infiltration of macrophages, and promoting the polarization of M2 macrophages, thereby reducing inflammatory damage, and protecting injury to the blood-brain barrier.

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.

Formation of Dispersible Taohong Siwu Tablets.

Here, we optimize the process used to formulate and prepare dispersible Taohong Siwu tablets and provide a foundation for expanding their clinical application. Taking dispersion uniformity and disintegration time as the indices for investigation, we used a single-factor test to match and filter the excipient categories for Taohong Siwu tablets. The formulation was optimized by an orthogonal test design. The content and dissolution rates of the effective substances in dispersible Taohong Siwu tablets when prepared with optimized prescriptions were determined by ultra-high performance liquid chromatography (UPLC), and the optimal preparation process was determined. The optimal composition for dispersible Taohong Siwu tablets was 17% Taohong Siwu extract powder, 1% magnesium stearate, 49% microcrystalline cellulose, 20% cross-linked polyvinylpyrrolidone, and 13% sodium carboxymethyl starch. When dispersible Taohong Siwu tablets were prepared by direct compression and the optimized prescription powder was uniformly dispersed within 3 min, the dissolution rate reached more than 90% within 50 min.When prepared according to the optimized methods,dispersible Taohong Siwu tablets disintegrate rapidly in water with good dispersion uniformity and controllable quality.

Taohong siwu decoction attenuates AIM2 and NLRC4 inflammasomes by ameliorates deoxyribonucleic acid damage after ischemic stroke.

Taohong siwu decoction (THSWD) has been shown to have a therapeutic effect on ischemic strokes (IS). However, it is not clear to us whether THSWD reduces deoxyribonucleic acid (DNA) damage after stroke and reduces the inflammatory response caused by the damage. Therefore, we constructed an IS model (I/R) in rats and performed oxygen-glucose deprivation/reoxygenation (OGD/R) on BV2 cells. Then ELISA, immunofluorescence staining, immunohistochemistry staining, and RT-qPCR were performed to detect the expressions of absent in melanoma 2 (AIM2), NLRC4, and Caspase-1 inflammasomes and other inflammatory factors. Experimental stroke causes DNA damage, and we found that the aforementioned inflammasomes as well as inflammatory factors were significantly inhibited after treatment with THSWD by comparing the model group with the model administration group. In addition, we examined the expression of AIM2, NLRC4, and Caspase-1 in BV2 cells of OGD/R and found that the expression of the aforementioned inflammasomes was significantly decreased in OGD/R by administration of THSWD-containing serum. Our data suggest that THSWD can reduced DNA damage after stroke as well as the inflammatory response caused by the damage.