Search

• The mechanism of Xingpi Capsule in the treatment of IBS-D was explored based on the integration analysis of three-omics.. • RNA sequencing combined with WB validation displayed that Xingpi Capsule exerted antidiarrheal effects by inhibiting the ASK1/P38 MAPK signaling pathway. • The inhibition of ASK1(Map3k5) may be ascribed to the increasing hypoxanthine after Xingpi Capsule treatment through RNA sequencing and LC-MS analysis. • 16S rRNA gene sequence analysis showed that Xingpi Capsule increased the content of hypoxanthine by reducing the abundance of Prevotella 9. Xingpi Capsule (XP), a commercially available over-the-counter herbal medicine in China, plays a prominent role in treating diarrhea-predominant irritable bowel syndrome (IBS-D). Nevertheless, the potential mechanisms remain unclear. This study aimed to investigate XP efficacy in IBS-D and elucidate the underlying molecular mechanisms. A rat IBS-D model was established by senna decoction gavage combined with restraint stress and swimming exhaustion. The changes in rat body weight and stool were recorded daily. Colon pathological changes and the number of colonic goblet cells of rats were observed by hematoxylin-eosin (HE) staining and Alcian blue plus periodic acid-Schiff (AB-PAS) staining, respectively. The expression of Occludin, a tight-junction-associated protein, was examined via immunohistochemistry. Images of colonic microvilli were obtained by TEM. Western blotting (WB) was used to analyze the protein expression of the ASK1/P38 MAPK pathway. The composition of the rat intestinal microbiota was detected by 16S rRNA sequencing. Changes in colonic metabolites were evaluated by liquid chromatography-mass spectrometry (LC-MS). Changes in colon RNA expression were assessed by RNA sequencing (RNA-Seq). The nontoxic range of hypoxanthine (HPX) was screened by Cell Counting Kit-8 (CCK8), the cell model of human colonic epithelial cells (NCM460) induced by lipopolysaccharide (LPS) was established, and the effective concentration of HPX was screened by CCK8. After transfection of pcDNA3.1-MAP3K5, Hoechst 33,342 staining, flow cytometry to detect cell apoptosis, and immunofluorescence to detect the fluorescence changes of ASK1 and ZO-1. WB detection of ASK1/P38 MAPK pathway protein expression changes. XP increased the body weight of IBS-D patients and reduced the loose stool rate, loose stool index, and Bristo score. In addition, XP mitigated colon lesions, increased the number of goblet cells and the expression of Occludin, and prevented severe distortion and effacement of the microvillous structure. Specifically, 16S rRNA gene sequence analysis showed that XP decreased the abundance of Desulfurium and Prevotella 9 at the phylum and genus levels while increasing the abundance of Bacteroides at the genus level. RNA-Seq combined with WB validation showed that XP exerted antidiarrheal effects by inhibiting the ASK1/P38 MAPK signaling pathway. Additionally, XP also increased the relative expression level of the metabolite HPX, as revealed by untargeted metabolomics analysis. Impressively, the correlation analysis between 16S rRNA sequencing and LC-MS suggested that HPX and Prevotella 9 are negatively correlated, which indicated that XP might increase the content of HPX by reducing the abundance of Prevotella 9. Meanwhile, a negative correlation between HPX and ASK1 was indicated through RNA-Seq and LC-MS, which suggested that the inhibition of ASK1 (Map3k5) may be ascribed to the increase in HPX after XP treatment. In vitro experiments have proven that HPX can alleviate LPS-induced NCM460 damage, specifically manifested as enhancing cell viability, reducing cell apoptosis, increasing ZO-1 expression, reducing the fluorescence intensity of MAP3K5 in the model group, and inhibiting the expression of ASK1/P38 MAPK pathway proteins. The protective effect of HPX was reversed after transfection with pcDNA 3.1-MAP3K5, which fully demonstrated that the protective mechanism of HPX was achieved by inhibiting MAP3K5 and its downstream pathways. XP displayed multifaceted protection against IBS-D in rats by regulating the intestinal microbiota, increasing the relative expression level of HPX, a metabolite of the microbiota, and inhibiting the ASK1/P38 MAPK signaling pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

• Neocryptotanshinone has a strong cardioprotective impact against myocardial ischemia/reperfusion damage. • Neocryptotanshinone promotes autolysosome degradation during aggrephagy. • Neocryptotanshinone up-regulates the ERK1/2-Nrf2-LAMP2 pathway. • Semi-synthesis of Neocryptotanshinone solves the problem of its high cost. Aggrephagy is a critical compensatory mechanism for the elimination of misfolded proteins resulting from stress and depends on the autolysosome degradation of protein aggregates. However, there have been few mechanism research related to aggrephagy in myocardial ischemia/reperfusion (I/R) injury. Neocryptotanshinone (NCTS) is a fat-soluble active compound extracted from Salvia miltiorrhiza , and may be cardioprotective against I/R. However, the efficacy and specific mechanism of NCTS on I/R have not been studied. The current study aimed to investigate the molecular mechanism of NCTS involved in the therapeutic effect on I/R, with a special emphasis on the up-regulation of the ERK1/2-Nrf2-LAMP2 pathway to increase autolysosomal degradation during aggrephagy. A rat model of myocardial I/R injury was constructed by left anterior descending (LAD) ligation-reperfusion. To verify cardiac protection, autolysosome clearance of protein aggregates, and their intracellular biological mechanism, an oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocyte model was created. NCTS was found to have a significant cardioprotective effect in I/R rats as evidenced by remarkably improved pathological anatomy, decreased myocardial damage indicators, and substantially enhanced cardiac performance. Mechanistically, NCTS might boost the levels of LAMP2 mRNA and protein, total and Ser40 phosphorylated Nrf2, and Thr202/Tyr204p-ERK1/2 protein. Simultaneously, the cytoplasmic Nrf2 level was reduced after NCTS administration, which was contrary to the total Nrf2 content. However, these beneficial changes were reversed by the co-administration with ERK1/2 inhibitor, PD98059. NCTS therapy up-regulated Rab7 protein content, Cathepsin B activity, and lysosomal acidity, while down-regulating autophagosome numbers, Ubiquitin (Ub), and autophagosome marker protein accumulations through the above signaling pathway. This might indicate that NCTS enhanced lysosomal fusion and hydrolytic capacity. It was also found that NCTS intervention limited oxidative stress and cellular apoptosis both in vivo and in vitro. We reported for the first time that NCTS promoted the autolysosome removal of protein aggregation both in vivo and in vitro , to exert the therapeutic advantages of myocardial I/R injury. This was reliant on the up-regulation of the ERK1/2-Nrf2-LAMP2 signaling pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

• Evaluated the mechanical behavior and fracture mechanism of red-bed mudstone under dry-wet cycling and prefabricated fracture planes with different loading angles. • Experimental results showed that increasing loading angles led to higher fracture loads, while more dry-wet cycles decreased fracture loads. • Dry-wet cycling caused the expansion of initial microcracks, forming weak layers that affected the main crack morphology and secondary crack distribution. • Acoustic emission count decreased with more dry-wet cycles, and rougher fracture surfaces were observed with increased cycles and loading angles. • Findings contribute to the understanding of red-bed mudstone stability and reliability under dry-wet cycling and prefabricated fracture planes with different loading angles. Red-bed mudstone is a common geological material in engineering construction in the Southwest region in China. Its initial fractures are easily influenced by the combined effects of dry-wet cycling and external loads. Therefore, evaluating the mechanical behavior and fracture mechanism of red-bed mudstone under dry-wet cycling and prefabricated fracture planes with different loading angles is of great significance. In this study, the cracked straight through Brazilian disc (CSTBD) specimens with a prefabricated crack were tested using an RMT testing machine. Multiple-scale analysis methods, including digital image correlation (DIC) and acoustic emission (AE) techniques, 3D scanning, and scanning electron microscopy (SEM), were employed to comprehensively analyze the mechanical evolution and fracture damage mechanism of mudstone under dry-wet cycling. The experimental results indicate that as the loading angle increases, the peak load gradually increases, while an increase in the number of dry-wet cycles leads to a decrease in peak load. At the same time, dry-wet cycling causes the expansion and connection of initial microcracks, forming weak layers that affect the morphology of the main crack and the distribution of secondary cracks. Furthermore, with an increasing number of dry-wet cycles, the acoustic emission count significantly decreases, and the relative high value range of acoustic emission count expands. 3D scanning results show that an increase in the number of dry-wet cycles results in rougher fracture planes, while an increase in loading angle generally increases the roughness of the fracture planes. Additionally, SEM observations reveal that with an increasing number of dry-wet cycles, intragranular fracture patterns and microcrack distribution become more widespread, and tensile and shear fracture patterns are also observed in the center of the fracture plane. These research findings have important practical value for evaluating the stability and reliability of red-bed mudstone under dry-wet cycling and prefabricated fracture planes with different loading angles. [ABSTRACT FROM AUTHOR]