CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

[Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway].

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

[Role of autophagy in liver injury induced by lung ischemia/ reperfusion in rats].

Objective: To investigate the liver injury induced by lung ischemia / reperfusion(LI/R) and the role of autophagy in its prevention and treatment. Methods: The lung ischemia/reperfusion injury(LI/RI) model was prepared by anesthetizing the rats, cutting the trachea for mechanical ventilation, and using an arterial clamp to close the pulmonary hilum to simulate the ischemic process, and releasing the arterial clamp after 30 min to resume perfusion for 3 h. SD rats(n=24)were randomly divided into sham operation(sham)group,ischemia/reperfusion(I/R)group,solvent(DMSO)group and autophagy inhibitor (3-MA) group, 6 rats in each group. The rats were intraperitoneally injected with medicine before operation. After the rat LI/RI model was established,the rats were killed, and the lung wet/dry weight ratio was used to evaluate the success of modeling, the venous blood was collected to measure the contents of ALT and AST, and the liver tissues were collected. Light and electron microscopes were used to observed the liver tissues and cell shapes. The protein and mRNA expression levels of autophagy related proteins were determined by Western blot and RT-qPCR to suggest autophagy levels. Results: Compared with sham group, the lung wet/dry weight ratios in other groups were elevated, and the liver tissues of other groups were damaged significantly. Serum levels of AST and ALT were increased significantly and liver tissue damage was obvious, especially in I/R group. The light microscopy showed that the arrangement of hepatic cords was disordered or broken, hepatic sinuses were dilated, and edema of liver cells were observed; transmission electron microscopy showed varying degrees of mitochondria swelling up in liver cells in the other groups. At the same time, the expressions of AMPK, Beclin 1 and LC3 mRNA were increased, but the expressions of mTOR and p62 mRNA were decreased; the protein expressions of p-AMPK, Beclin 1, LC3-B were increased significantly, but those of p-mTOR and p62 were decreased (all P＜0.05). Compared with DMSO group, the injury of liver tissue in 3-MA group was alleviated, the damage degree of mitochondrial ultrastructure was lower, the levels of AST and ALT were decreased, the transcription and protein expression levels of autophagy related protein in liver tissue were decreased (P＜0.05). However, the injury degree of IR and DMSO groups were similar, and there was no significant differences in each index (P＞0.05). Conclusion: Lung ischemia/reperfusion can cause liver injury in rats. Autophagy can mediate liver injury induced by lung ischemia / reperfusion in rats and inhibiting autophagy can effectively reduce liver injury induced by LI/R in rats.

[Effects of panax notoginseng saponins on pulmonary vascular remodeling in rats with pulmonary hypertension and its mechanisms].

Objective: To investigate the effects of panax notoginseng saponins (PNS) on pulmonary vascular remodeling and SIRT1/FOXO3a/p27 pathway in pulmonary arterial hypertension (PAH) rats. Methods: Male SD rats weighing 200~250g were randomly divided into control group, monocrotaline group (MCT) and monocrotaline + panax notoginseng saponins group (MCT+PNS), with 10 rats in each group. The rats in control group were injected intraperitoneally with normal saline 3 ml/kg on the first day, then injected intraperitoneally with normal saline 2.5 ml/kg every day. The rats in MCT group were injected intraperitoneally with MCT 60 mg/kg on the first day, followed by daily injection of normal saline 2.5 ml/kg. In MCT+PNS group, 60 mg/kg MCT was injected intraperitoneally on the first day, and 50 mg/kg PNS was injected intraperitoneally every day. The above models were fed conventionally for 4 weeks. After the modeling was completed, the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) of rats in each group were detected by right heart catheter method, weighed and calculated right ventricular hypertrophy index (RVHI), and the pulmonary vascular structure and morphological changes were observed by HE and Masson staining. The protein and gene expressions of SIRT1, FOXO3a, p27, PCNA and Caspase-3 were detected by qPCR and Western blot. Results: Compared with control group, mPAP, RVSP and RVHI in MCT group were increased significantly (P＜0.01), pulmonary vessels were thickened significantly and collagen fibers were increased, protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were decreased (P＜0.05 or P＜0.01). The protein and gene expressions of PCNA were increased (P＜0.05). Compared with MCT group, the levels of mPAP, RVSP and RVHI in MCT+PNS group were decreased significantly (P＜0.05 or P＜0.01), pulmonary vascular thickening was alleviated and collagen fibers were reduced. The protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were increased (P＜0.05 or P＜0.01), while the protein and gene expressions of PCNA were decreased (P＜0.05 or P＜0.01). Conclusion: Panax notoginseng saponins can relieve pulmonary vascular remodeling in rats with pulmonary hypertension by activating SIRT1/FOXO3a/p27 pathway.

Impact of Low-Dose rATG Prior to Matched Sibling Donor Hematopoietic Stem Cell Transplantation for Hematologic Malignancies: Reduced Risk of Chronic Graft-versus-Host Disease and Improved Survival Outcomes.

PURPOSE: To explore the efficacy of low-dose rabbit antithymocyte globulin (rATG) in matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) for patients with acute leukemia or myelodysplastic syndrome. PATIENTS AND METHODS: We performed a retrospective study of 79 patients with hematologic malignancies who received MSD-HSCT. All patients received standard graft-versus-host disease (GVHD) prophylaxis comprising cyclosporine, mycophenolate mofetil and short-term methotrexate. Among them, 38 were administered 5 mg/kg rATG as part of GVHD prophylaxis. Clinical outcomes including overall survival (OS), GVHD and relapse were analyzed. RESULTS: No graft failure occurred in the antithymocyte globulin (ATG) or non-ATG group. The cumulative incidences of grade 2-4 and 3-4 acute GVHD at day +100 were 13.3% versus 19.5% (p=0.507) and 5.7% versus 15.2% (p=0.196), respectively. The 2-year cumulative incidences of chronic GVHD (cGVHD) were 35.4% and 60.4% (p=0.039), and those of extensive cGVHD were 12.9% and 40.0% (p=0.015), respectively. In a multivariate analysis, the use of low-dose rATG was an independent protective factor for extensive cGVHD (hazard ratio [HR] 0.256; 95% confidence interval [CI], 0.080 to 0.822, p=0.022). The 2-year OS was 88.1% and 68.4% (p=0.038), respectively, and the use of low-dose rATG was the only protective factor in the multivariate analysis (HR 0.216; 95% CI, 0.059 to 0.792, p=0.021). There was no significant difference between the two groups in terms of the 2-year cumulative incidence of relapse, leukemia-free survival or GVHD-free and relapse-free survival. CONCLUSION: Low-dose rATG used in MSD-HSCT as part of the conditioning regimen results in a reduced incidence of cGVHD and improves survival outcomes.

[A genetic sub-structure study of the Tibetan population in Southwest China].

Tibetan is a typical ethnic minority population in Southwest China, which can be divided into U-Tsang, Kham, Amdo, Jiarong and other sub-populations. However, the genetic structure of these sub-populations has not been comprehensively analyzed, especially from the perspective of paternal and maternal lineages. Based on genetic markers of autosomes, the Y chromosome and mitochondria, we studied four Tibetan populations (the U-Tsang population in Tibet Autonomous Region; the Kham population in Garze, Sichuan province; the Amdo population in Qinghai province and the Jiarong population in Aba, Sichuan province) to interpret their genetic structure. The mini-sequencing technology was used to detect the genotype of each maker. Meanwhile, the PowerPlex ?Y23 and DNA Typer TM Y26 kit were applied to genotype Y-STRs. Subsequently, the genetic structure was analyzed by heatmap and principal component analysis, ancestry component, haplogroup frequency, network map and multi-dimensional scaling analysis. The results showed that the four Tibetan populations could be divided into three sets based on the autosomal and Y-chromosomal genetic markers, in which set 1 was the U-Tsang population in the Tibetan Plateau, set 2 comprised of the Kham and Amdo populations in the surrounding areas of the plateau, and set 3 was the Jiarong population that resided in the Tibetan and Yi Corridor. No significant difference was observed in mitochondrial genetic markers among four Tibetan populations. In general, multi-category genetic information provides a new comprehensive insight into the Tibetan sub-population.