Study on the function of Huazhuo Jiedu Decoction in promoting the homing of bone marrow mesenchymal stem cells and contributing to the treatment of ulcerative colitis.

Objective: To study the function of Huazhuo Jiedu Decoction (HZJD) in promoting the homing of bone marrow mesenchymal stem cells (BMSCs) and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis. Methods: Bone mesenchymal stem cells derived from mice were isolated and cultured, osteogenic and adipogenic assays to study the differentiation ability of BMSCs, and flow cytometry was used to detect the surface marker of the third generation cells. 30 mice were selected and divided into blank group, model group, HZJD group, BMSCs group, and HZJD combined with BMSCs group. Mouse colon length, body weight, and DAI score were used to assess efficacy. The levels of IL-6, IL-1ß, TNF-α, and IFN-γ in serum were measured by ELISA. BMSCs transfected with GFP were used to mark the homing of BMSCs in mice. The BMSCs tagging protein CD90+/CD29+ was detected by immunofluorescence. H&E staining detects damage to the colon and the inflammatory response. The expression levels of claudin-2, claudin-4, occludin, and ZO-1 in colon tissues were detected by Western blot. Results: After subculture, the cell grew with adherence. Flow cytometry showed that the cells were CD73+/CD90+/CD29+/CD45-/CD34-, which belonged to bone mesenchymal stem cells. ELISA showed that the treatment with HZJD and BMSCs suppressed the DSS-induced inflammatory response. BMSCs carrying GFP can be detected in intestinal tissues. Immunofluorescence showed that the HZJD combined with the BMSCs group had more BMSCs homing to the colonic tissue. The results of H&E and Western blot showed that DSS-induced intestinal mucosal damage in UC mice was repaired by HZJD and BMSCs, and the abnormal tight junction proteins claudin-2, claudin-4, occludin, and ZO-1 were normalized. Conclusion: HZJD has a therapeutic effect on ulcerative colitis by promoting the migration of BMSCs to ulcers of the colon and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis.

Diosmetin alleviated cerebral ischemia/reperfusion injury in vivo and in vitro by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway.

Cerebral ischemia/reperfusion (I/R) injury is caused by blood flow recovery after an ischemic stroke, and effective treatments targeting I/R injury are still insufficient. Oxidative stress is known to play a pivotal role in the pathogenesis of cerebral I/R injury. Previous studies have revealed that diosmetin could protect against oxidative stress in cerebral I/R injury, but the underlying mechanisms have not been fully revealed. The present study was undertaken to investigate the effects and mechanisms of action of diosmetin on cerebral I/R injury. In vivo, rats were orally gavaged with diosmetin for seven days, and middle cerebral artery occlusion (MCAO) was established to simulate cerebral I/R injury. The neurological deficit score, cerebral infarct volume, and cortical pathological lesions were measured. In vitro, PC12 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). To clarify the mechanism, the SIRT1 inhibitor EX527 and the small interfering RNA (siRNA) of SIRT1 were used to downregulate the SIRT1 protein level, respectively. The contents of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) were determined with commercial kits. The protein expressions of SIRT1, total Nrf2 (T-Nrf2), nucleus Nrf2 (N-Nrf2), NQO1 and HO-1 were measured by western blotting. The results showed that diosmetin pretreatment improved neurological outcomes, decreased the cerebral infarct volume and pathological lesions, and inhibited oxidative stress in cerebral I/R rats. In PC12 cells, diosmetin increased cell viability, reduced lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) level, and inhibited oxidative stress. Besides, diosmetin increased the protein expressions of SIRT1, T-Nrf2, N-Nrf2, NQO1 and HO-1 both in vivo and in vitro. However, administration of EX527 or silencing the SIRT1 gene with its siRNA eliminated the beneficial effects of diosmetin. Meanwhile, inhibition of SIRT1 decreased the levels of Nrf2 and the protein expressions of its downstream antioxidants NQO1 and HO-1. In conclusion, our data suggested that diosmetin could attenuate cerebral I/R injury by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway.

In Silico and In Vivo Studies on the Mechanisms of Chinese Medicine Formula (Gegen Qinlian Decoction) in the Treatment of Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease, and Gegen Qinlian Decoction (GQD), a Chinese botanical formula, has exhibited beneficial efficacy against UC. However, the mechanisms underlying the effect of GQD still remain to be elucidated. In this study, network pharmacology approach and molecular docking in silico were applied to uncover the potential multicomponent synergetic effect and molecular mechanisms. The targets of ingredients in GQD were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of TCM (BATMAN-TCM) database, while the UC targets were retrieved from Genecards, therapeutic target database (TTD) and Online Mendelian Inheritance in Man (OMIM) database. The topological parameters of Protein-Protein Interaction (PPI) data were used to screen the hub targets in the network. The possible mechanisms were investigated with gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was used to verify the binding affinity between the active compounds and hub targets. Network pharmacology analysis successfully identified 77 candidate compounds and 56 potential targets. The targets were further mapped to 20 related pathways to construct a compound-target-pathway network and an integrated network of GQD treating UC. Among these pathways, PI3K-AKT, HIF-1, VEGF, Ras, and TNF signaling pathways may exert important effects in the treatment of UC via inflammation suppression and anti-carcinogenesis. In the animal experiment, treatment with GQD and sulfasalazine (SASP) both ameliorated inflammation in UC. The proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) induced by UC were significantly decreased by GQD and SASP. Moreover, the protein expression of EGFR, PI3K, and phosphorylation of AKT were reduced after GQD and SASP treatment, and there was no significance between the GQD group and SASP group. Our study systematically dissected the molecular mechanisms of GQD on the treatment of UC using network pharmacology, as well as uncovered the therapeutic effects of GQD against UC through ameliorating inflammation via downregulating EGFR/PI3K/AKT signaling pathway and the pro-inflammatory cytokines such as TNF-α, IL-1ß and IL-6.

[Pyroptosis induced by zinc oxide nanoparticles in A549 cells].

OBJECTIVE: To explore whether exposure to zinc oxide nanoparticles (ZnO-NPs) induced pyroptosis in human alveolar type II epithelial cells (A549). METHODS: A549 cells were stimulated with different concentrations of ZnO-NPs (10 and 20 microg/ml) for 8 and 24h, respectively. The activity of lactic dehydrogenase (LDH) and the levels of interleukin-1beta (IL-1beta) in cell culture media were respectively measured by LDH activity kit and human IL-1beta ELISA kit. The activity of caspase-1 in A549 cells was detected by caspase-1 colorimetric assay kit. RESULTS: At 8h exposure to ZnO-NPs, there was no significant difference in the activity of LDH in the cell culture media among the ZnO-NPs-treated and control groups, but the activity of caspase-1 and the levels of IL-1beta in A549 cells were significantly increased in 20 microg/ml ZnO-NPs group compared to that in the control group (P < 0.05). Levels of IL-1beta and activity of LDH in the groups treated with ZnO-NPs (10 or 20 microg/ml) were significantly higher than that in the control group after 24 h exposure to ZnO-NPs, but there was no significant difference in the activity of caspase-1 among ZnO-NPs and control group. CONCLUSION: ZnO-NPs (20 microg/ml) treatment induces early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in A549 cells.

[Coal tar pitch smoke extract-induced pyroptosis in human bronchial epithelial cells].

OBJECTIVE: To explore whether coal tar pitch smoke extract (CTP) induced pyroptosis in human bronchial epithelial cells (BEAS-2B). METHODS: BEAS-2B cells were treated with different concentrations of CTP (1, 3 µg/ml) for 8h and 24 h, respectively. Lactic dehydrogenase (LDH) activity and interleukin-1 beta (IL-1ß) levels in the supernatants of cell culture media were measured with LDH activity or human IL-1ß ELISA kit, respectively. The activity of Caspase-1 was measured with Caspase-1 colorimetric assay kit. RESULTS: The activity of caspase-1 in 1 and 3 µg/ml CTP groups were (9.29 ± 0.30) and (8.67 ± 0.59) µmol/ml respectively which were both significantly increased compared to that (7.42 ± 0.59) µmol/ml in the control group (P < 0.05) after 8 h exposure, but there was no significant difference in the activity of LDH and levels of IL-1ß in the cell culture media among the CTP and control groups. 24 h after exposure, the activity of LDH in the CTP (1, 3 µg/ml) groups were (1323.03 ± 28.53) and (1148.45 ± 16.42) U/dl respectively which were significantly higher than that (1091.93 ± 26.64) U/dl in the control group (P < 0.05), and the levels of IL-1ß in the CTP (1 and 3 µg/ml) groups were (125.37 ± 25.00) pg/ml and (92.04 ± 19.09) pg/ml respectively which were significantly higher than that (46.20 ± 14.43) pg/ml in the control group (P < 0.05), but there was no significant difference in the activity of Caspase-1 among CTP and control groups (P < 0.05). CONCLUSION: CTP treatment induced early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in human bronchial epithelial cells.