Nan fang yi ke da xue xue bao = Journal of Southern Medical University最新文献

Objective: To explore the effects of Qingshen Granules (QSG) on adenine-induced renal fibrosis in mice and in uric acid (UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes, miR-330-3p and CREBBP.

Methods: A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg^-1·d^-1 via gavage for 12 weeks. An adenoassociated virus vector was injected into the tail vein, and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9, Hsp70, and TSG101 and expressions of Col-III, α-SMA, FN, and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining. In the cell experiment, NRK-49F cells were stimulated with uric acid (400 μmol/L) followed by treatment with QSG-medicated serum from SD rats, and the changes in expressions of the exosomal markers and Col-III, α-SMA, FN, and E-cad were analyzed. Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP, whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells.

Results: The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9, Hsp70, and TSG101, which were decreased by treatment with QSG. The expressions of Col-III, α-SMA, and FN increased and Ecad decreased in the mouse models but these changes were reversed by QSG treatment. QSG treatment obviously alleviated renal fibrosis in the mouse models. Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p, increased CREBBP levels, and reduced fibrosis in the mouse models. Dual luciferase assay confirmed CREBBP as a target of miR-330-3p, which was consistent with the results of the cell experiments.

Conclusion: QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.

Objective: To investigate the effect of sanguinarine (SAN) on proliferation and ferroptosis of colorectal cancer cells.

Methods: SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC₅₀ of SAN in the two cells. The inhibitory effects of SAN on proliferation, invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays. ROS production in the treated cells was analyzed with flow cytometry, and lipid peroxide production was assessed by detecting malondialdehyde (MDA) level. Glutathione (GSH) levels in the cells were detected, and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4.

Results: SAN significantly inhibited the proliferation, invasion and migration of SW620 and HCT-116 cells. SAN treatment significantly promoted ROS production, increased intracellular MDA level, and lowered GSH level in the two cells (P<0.05). Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4 (P<0.05).

Conclusion: SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4, which may serve as a new therapeutic target for colorectal cancer.

Objective: To explore the causal relationship between sleep phenotype and idiopathic normal pressure hydrocephalus (iNPH) using two-sample bidirectional Mendelian randomization.

Methods: The exposure data including 8 sleep phenotypes used in this study were obtained from GWAS catalog, FinnGenR10 and MRCIEU GWAS. The outcome data for idiopathic normal-pressure hydrocephalus were obtained from FinnGen R10. We used the inverse-variance weighted (IVW) method to perform the principal analyses. Cochrane Q-statistics test was used to assess the heterogeneity and MR Egger‑intercept test performed to evaluate the pleiotropy for sensitivity analyses.

Results: IVW result showed that frequent daytime nap was associated with higher odds of iNPH (OR=3.3393, 95 CI% : 1.0646-10.4742, P=0.0270). Cochrane Q-statistics test and MR Egger‑intercept test showed that the MR analysis had no pleiotropy or heterogeneity (P > 0.05). The external validation reproduced this result (OR=2.5660, 95 CI% : 1.1680-5.6373, P=0.0189; OR=4.0424, 95 CI% : 1.5709-10.4024, P=0.0038). Reverse Mendelian randomization suggested that iNPH did not have significant impact on sleep phenotype.

Conclusion: The frequency of daytime naps is causally associated with iNPH, and reducing the frequency of weekly daytime naps can reduce the risk of iNPH in the elderly population.

Objective: To investigate the effect of dihydroartemisinin (DHA) for enhancing the inhibitory effect of cisplatin (DDP) on DDP-resistant nasopharyngeal carcinoma cell line HNE1/DDP and explore the mechanism.

Methods: CCK-8 method was used to assess the survival rate of HNE1/DDP cells treated with DHA (0, 5, 10, 20, 40, 80, and 160 μmol/L) and DDP (0, 4, 8, 16, 32, 64, 128 μmol/L) for 24 or 48 h, and the combination index of DHA and DDP was calculated using Compusyn software. HNE1/DDP cells treated with DHA, DDP, or their combination for 24 h were examined for cell viability, proliferation and colony formation ability using CCK-8, EdU and colony-forming assays. Flow cytometry was used to detect cell apoptosis and intracellular reactive oxygen species (ROS). The expression levels of apoptosis-related proteins cleaved PARP, cleaved caspase-9 and cleaved caspase-3 were detected by Western blotting. The effects of N-acetyl-cysteine (a ROS inhibitor) on proliferation and apoptosis of HNE1/DDP cells with combined treatment with DHA and DDP were analyzed.

Results: Different concentrations of DHA and DDP alone both significantly inhibited the viability of HNE1/DDP cells. The combination index of DHA (5 μmol/L) combined with DDP (8, 16, 32, 64, 128 μmol/L) were all below 1. Compared with DHA or DDP alone, their combined treatment more potently decreased the cell viability, colony-forming ability and the number of EdU-positive cells, and significantly increased the apoptotic rate, intracellular ROS level, and the expression levels of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 in HNE1/DDP cells. N-acetyl-cysteine pretreatment obviously attenuated the inhibitory effect on proliferation and apoptosis-inducing effect of DHA combined with DDP in HNE1/DDP cells (P<0.01).

Conclusion: DHA enhances the growth-inhibitory and apoptosis-inducing effect of DDP on HNE1/DDP cells possibly by promoting accumulation of intracellular ROS.

Objective: To explore the correlation of baseline CCL19⁺ dendritic cell (CCL19⁺ DC) infiltration in lung adenocarcinoma microenvironment with immunotherapy efficacy and CD8⁺ T cell infiltration.

Methods: We retrospectively analyzed the data of patients with lung adenocarcinoma hospitalized at First Affiliated Hospital of Henan University of Science and Technology from January, 2020 to December, 2023, and collected tissue samples from 96 patients undergoing immunotherapy for assessing CCL19⁺ DC and CD8⁺ T cell infiltration using immunofluorescence assay. We evaluated the predictive value of baseline CCL19⁺ DCs for patient responses to immunotherapy using receiver-operating characteristics (ROC) curves and analyzed the correlations of baseline CCL19⁺ DC expression with immunotherapy efficacy and CD8⁺ T cell and cytotoxic T lymphocyte (CTL) infiltrations. In co-culture systems of lung adenocarcinoma PC9 cells, CD8⁺ T cells and DCs (overexpressing CCL19 with or without anti PD-1 antibody treatment), the expressions of granzyme B, perforin, IFN-γ, and Ki-67 in T cells were analyzed using flow cytometry.

Results: The patients with partial or complete remission following immunotherapy had a significantly higher baseline CCL19⁺ DC infiltration level in lung adenocarcinoma tissues than those with poor responses. CCL19⁺ DC infiltration had an area under ROC curve of 0.785, a sensitivity of 75.6%, and a specificity of 62.8% for predicting immunotherapy efficacy. The expression of CD8⁺ T cell surface molecules Granzyme B (P<0.01), Perforin (P<0.01), IFN-γ (P<0.01) and Ki-67 (P<0.001) in patients with high expression of CCL19⁺ DC were higher than those in patients with low expression of CCL19⁺ DC. The baseline CCL19⁺ DC infiltration level was positively correlated with immunotherapy efficacy (P=0.003), CTL infiltration of (r=0.6657, P<0.001) and CD8⁺ T cell infiltration (P=0.007). In the co-cultured cells, CCL19 overexpression combined with anti-PD1 treatment of the DCs more strongly enhanced cytotoxicity and proliferation of CD8⁺ T lymphocytes than either of the single treatments (P<0.01 or 0.001).

Conclusion: The baseline CCL19⁺ DC infiltration level in lung adenocarcinoma microenvironment is positively correlated with immunotherapy efficacy and CTL infiltration and can thus predict the response to immunotherapy.

Objective: To explore the inhibitory effect ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on growth of glioblastoma (GBM) and the underlying mechanism.

Methods: We analyzed LSD1 expressions in GBM and normal brain tissues based on data from TCGA and HPA databases. Female BALB/c mouse models bearing xenografts derived from U87 cells or cells with lentivirus-mediated LSD1 silencing or Notch overexpression were treated with saline or 400 µg/kg ORY-1001 by gavage every 7 days, and GBM formation and survival time of the mice were recorded. The effect of ORY-1001 on GBM cell viability was assessed, and its effect on LSD1 expression was analyzed with Western blotting. The genes and pathways associated with LSD1 were analyzed using bioinformatics methods. Western blotting and qRT-PCR were used to detect Notch/HES1 pathway expression after LSD1 silencing and ORY-1001 treatment. The impact of ORY-1001 on viability of U87 cells with Notch1 silencing or overexpression was assessed, and the regulatory effects of ORY-1001 on Notch/HES1 pathway were analyzed using chromatin immunoprecipitation assay.

Results: A high expression of LSD1 in GBM was negatively correlated with patient survival (P < 0.001). ORY-1001 and LSD1 silencing obviously reduced tumor burden and prolonged the survival time of GBM-bearing mice. ORY-1001 treatment significantly inhibited the viability and dose-dependently decreased LSD1 expression in GBM cells, and such inhibitory effect of ORY-1001 was attenuated by LSD1 silencing. The Notch pathway enriched the differential genes related to LSD1, and Notch/HES1 pathway expression was significantly down-regulated after LSD1 silencing and ORY-1001 treatment. Notch1 overexpression significantly attenuated the anti-tumor effect of ORY-1001 on GBM. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and the Notch pathway target genes including Notch3, HES1 and CR2.

Conclusion: ORY-1001 down-regulates the Notch/HES1 pathway by inhibiting LSD1 expression to suppress the growth of GBM in mice.

Objective: To investigate the mechanism of sanguinarine (SA) for alleviating ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) in mice.

Methods: Male C57BL/6 mouse models of 3.5% DSS-induced UC were randomized for treatment with 1, 5 and 10 mg/kg SA by gavage, 400 mg/kg sulfasalazine by gavage, or 10 mg/kg SA combined with intraperitoneal injection of 30 mg/kg ML385 (a Nrf2 inhibitor). The changes in intestinal inflammation was assessed by monitoring weight changes, disease activity index (DAI) score, colon length measurement, and HE staining. After the treatments, the colon tissues were collected for detection of malondialdehyde (MDA) content using colorimetry, mRNA expressions of inflammatory factors using RT-qPCR, and the expressions of Nrf2, HO-1, Keap-1, p-p65, p65, occludin, and ZO-1 proteins were detected using Western blotting.

Results: SA treatment obviously alleviated weight loss, colon length shortening and DAI score increase and ameliorated structural destruction of the colon glands and colonic crypts in mice with DSSinduced UC. SA intervention significantly decreased the levels of TNF-α, IL-1β and IL-6 mRNA and lowered ROS and MDA levels in the colon tissue of UC mice. The mouse models receiving SA treatment showed significantly increased expressions of Nrf2, HO-1, occludin and ZO-1 and lowered expressions of Keap-1 and P-P65 in the colon tissue without significant changes of p65 expression, and these changes were SA dose-dependent. Treatment with ML385 obviously attenuated the effect of highdose SA for improving UC in the mouse models.

Conclusion: SA can improve UC-like enteritis in mice possibly by activating the Nrf2 pathway and inhibiting the NF-κB pathway in the colon tissue.

Objective: To investigate the effects of Huangqin Qingrechubi Capsule (HQC) on inflammation and uric acid and lipid metabolism in rats with gouty arthritis (GA) and its mechanism.

Methods: SD rat models of GA established by injecting monosodium urate into the right ankle joint were treated with saline, colchicine and HQC at low, medium and high doses (n=10) by gavage for 7 days. Toe swelling of the rats was detected at 4, 8, 24, 48 and 72 h after modeling, and synovial histological changes were observed with HE staining. Serum levels of interleukin-10 (IL-10), IL-18, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), adiponectin, leptin, resistin and visfatin were measured by ELISA, and the levels of high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), total cholesterol (TC), and uric acid (BUA) were detected. RTqPCR and Western blotting were used to detect the mRNA expressions of phosphatase and tensin homolog (PTEN), phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) and the protein expressions of PTEN, PI3K, p-PI3K, AKT and p-AKT.

Results: The rat models of GA showed obvious toe swelling, which reached the peak level at 48 h. HE staining revealed massive inflammatory cell infiltration and synovial tissue hyperplasia. The rat models showed significantly increased expressions of TNF-α, TGF-β1, IL-18, TC, TG, leptin, resistin and visfatin, BUA, p-PI3K, and p-AKT and lowered levels of IL-10, APN, HDL-C, and PTEN. Treatment with HQC and colchicine obviously improved these changes and alleviated synovial pathologies and toe swelling in the rat models.

Conclusion: HQC can improve inflammation and correct the imbalance of uric acid and lipid metabolism in GA rats possibly by inhibiting the PTEN/PI3K/AKT signaling pathway.

Objective: To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice.

Methods: PF4-Cre and Rictor^fl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy.

Results: Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice.

Conclusion: Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Objective: To investigate the mechanism by which conbercept reverses transforming growth factor-β₂ (TGF-β₂)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs).

Methods: Cultured HLEC SRA01/04 cells were treated with TGF-β₂, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway.

Results: Conbercept significantly reduced TGF-β₂-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-β₂+conbercept group than in TGF-β₂ group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β₂-induced EMT (P <0.01).

Conclusion: Conbercept inhibits TGF-β₂ induced EMT by downregulating the expression of pSmad2/3 in TGF-β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.