To investigate the role of RAS-like activator 2 (RASAL2) in metabolic dysfunction-associated steatohepatitis (MASH) and its underlying mechanism.

A murine MASH model was established by feeding a high-fat diet for 24 weeks. Hepatic inflammatory gene expression levels were quantified by qRT-PCR. Hepatic immune cell infiltration was assessed by flow cytometry. Bone marrowderived macrophages (BMDMs) were isolated and cultured in vitro, followed by stimulation with lipopolysaccharide to induce M1 polarization. The expression levels of M1 polarization markers (Nos2, Il1b, Il6, Tnf, Arg1, CD206, Il10) were measured by qRT-PCR. The protein expressions of p-IκBα, IκBα, p-IKKβ, IKKβ, p-P65, and P65 were determined by Western blot.

Compared with wild-type littermates, RASAL2-knockout mice exhibited markedly decreased hepatic inflammatory cell infiltration, reduced hepatic mRNA levels of Il1b, Il6, and Tnf, and increased Il10 expression. Flow cytometry revealed a reduction in hepatic immune cells infiltration and a decrease in M1 macrophages in RASAL2-knockout mice. In vitro experiments showed that RASAL2 knockout reduced the expression of M1 polarization marker genes (Nos2, Il1b, Il6, Tnf), increased the expression of M2 polarization marker genes (Arg1, CD206, Il10), and inhibited the activation of NF-κB pathway.

RASAL2 knockout attenuates hepatic inflammatory injury in MASH and inhibits M1 macrophage polarization. Its mechanism may be related to the suppression of the NF-κB signaling pathway.

To investigate the interventional effects of the Bu-fei Yi-shen Formula(BYF)on airway mitochondrial damage in rats with stable chronic obstructive pulmonary disease (COPD).

Forty rats were randomly assigned to four groups: Control, COPD model, BYF, and Doxofylline (DOX). From week 1 to week 8, stable COPD was induced by twice-daily exposure to cigarette smoke (30 minutes each time) combined with intranasal instillation of Klebsiella pneumoniae (once weekly). From week 9 to week 16, the control and COPD model groups were administered normal saline by gavage at a dose of 2 mL per rat. The BYF and DOX groups received 2 mL of BYF or DOX, respectively, by gavage. In week 16, tissue samples were collected. Subsequently, the degree of mitochondrial damage in lung tissues of COPD rats was evaluated from multiple dimensions, including pulmonary function, histopathology and ultrastructure of lung tissue, mitochondrial membrane potential, activities of mitochondrial respiratory chain complexes, and mRNA and protein expression levels of IP3R, GRP75, and VDAC1.

Compared with the control group, the COPD group showed significantly lower pulmonary function indices(P < 0.05, P < 0.01). Histopathological examination revealed typical COPD-related alterations, including alveolar rarefaction, rupture and fusion of alveolar walls, thickening and corrugation of bronchial walls, accompanied by extensive inflammatory cell infiltration. The mitochondrial membrane potential in lung tissues of the COPD group was significantly reduced, and the activities of respiratory chain complexes II and IV decreased(P < 0.05). Serum levels of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), transforming growth factor- β1(TGF-β1), and interleukin-6(IL-6)were significantly elevated(P < 0.05, P < 0.01). The mRNA and protein expression levels of IP3R, GRP75, and VDAC1 in lung tissues were significantly elevated in the COPD group compared with the control group(P < 0.05, P < 0.01). These pathological alterations were alleviated to varying degrees in the treatment groups, with the BYF group demonstrating greater improvement than the DOX group.

BYF alleviates chronic inflammation and mitochondrial damage in rats with COPD, and its underlying mechanism may be associated with the inhibition of the IP3R/GRP75/VDAC1 signaling pathway.

To construct T cell receptor β chain(TCRβ) gene knockout mouse model using CRISPR/Cas9 technology for genotype analysis and preliminary phenotypic characterization.

Tcrb^-/- F0 generation mice were obtained by using CRISPR/Cas9 gene targeted knockout technology followed by embryo transfer. F1 generation mice were subsequently produced by mating sexually mature F0 Tcrb^-/- mice with wild-type littermates. Genotypes were determined by polymerase chain reaction analysis of mouse tails. These results at the protein and cellular levels were validated using Western blot and flow cytometry, respectively.

The Tcrb gene knockout mice were successfully constructed and three genetically stable variants were obtained: wild-type(Tcrb^+/+), heterozygous(Tcrb^+/-), and homozygous knockout (Tcrb^-/-) founder lines. Western blot analysis showed that TCRβ protein expression was barely detectable in the tissues of Tcrb^-/- mice. Flow cytometry results indicated that compared with Tcrb^+/+ mice, the numbers of CD4⁺ and CD8⁺ T cells in the peripheral blood, thymus and spleen of Tcrb^+/- mice were significantly reduced, while in Tcrb^-/- mice, CD4⁺ and CD8⁺ T cells were almost not expressed and the CD4⁺/CD8⁺ ratio was significantly reduced.

The Tcrb^-/- mice were successfully constructed, which provides an experimental animal model for in-depth exploration of the pathological mechanisms of autoimmune diseases and the identification of potential drug targets.

To explore the mechanism of action of quercetin in the treatment of septic cardiomyopathy using network pharmacology and molecular docking techniques, and to validate the predicted results by in vivo experiments.

Drug targets of quercetin and targets of septic cardiomyopathy were searched through database, and the intersection of the two was taken. A protein-protein interaction network map was constructed, and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to enrich the analysis of the intersection targets, and molecular docking was performed on the core targets of key pathways. C57BL/6J male mice were randomly divided into four groups: sham surgery group (sham group), sepsis group, high-dose quercetin group, and low-dose quercetin group. Cardiac function was evaluated using small animal cardiac ultrasound, serum myocardial enzymes and troponin were detected, the changes of the pathology and ultra-structure in myocardial tissue were observed by HE staining and transmission electron microscopy, and the expression of core targets was verified by RT-PCR and Western blot.

A total of 98 intersection genes related to disease targets of quercetin were obtained, and 194 biological processes, 15 cell components, and 26 molecular functions were obtained by GO analysis. KEGG pathway analysis found that quercetin was most likely to treat septic cardiomyopathy through the IL-17 signaling pathway, and molecular docking showed that matrix metalloproteinase-9(MMP-9)and interleukin-1β (IL-1β)had the lowest binding energy. Quercetin significantly improved cardiac function in mice with septic cardiomyopathy, markedly reduced myocardial enzymes and troponin, apparently reduced myocardial cell edema and inflammatory cell infiltration, and improved myocardial sarcomere disorder and mitochondrial swelling. PCR and Western blot results showed that quercetin reduced the levels of myocardial IL-17, TRAF6, MMP9 and IL-1β in mice with septic cardiomyopathy, verifying the prediction results of network pharmacology and molecular docking.

Quercetin may alleviate myocardial injury in sepsis by inhibiting the IL-17 pathway.

To investigate the mechanism of Piezo1 activation participating in rat coronary artery smooth muscle cells (CASMCs) calcification under high calcium-phosphate stimulation.

CASMCs were treated with β -glycerophosphate and CaCl₂ for 10 days to induce calcification, and cells were divided into the general group(GM)and the calcification group (CM). Then, CASMCs were intervened with inhibitor GsMTx4, siRNA-Piezo1 and agonist Yoda1, respectively. The calcification of CASMCs was examined via alizarin red staining, and the expression of proteins associated with phenotype transition and mitochondrial dynamics was examined via Western blot.

Compared with the GM, the CM had more obvious calcification(P < 0.01). The expression of Piezo1, Runx2, BMP2 and OPN increased(P < 0.01), and the expression of SM22α and SM-MHC decreased(P < 0.01)in the CM. After intervention with GsMTx4 and siRNA-Piezo1, calcification was significantly alleviated(P < 0.01), while intervention with Yoda1 enhanced calcification(P < 0.01). In addition, when stimulated with high calciumphosphorus, there was an increase in the expression of DRP1^Ser616 and FIS1(P < 0.01), and a decrease in the expression of MFN1 and MFN2(P < 0.01). Intervention with Yoda1 could aggravate mitochondrial fission (P < 0.01), and intervention with Mdivi-1 could alleviate CASMCs calcification (P < 0.01).

Piezo1 activation can mediate the calcification of CASMCs under high calcium and phosphorus stimulation, and its mechanism may be related to the induction of mitochondrial dynamics imbalance.