2024, 34(6):11-17.DOI: 10.3969/j.issn.1671-7856.2024.06.002
Abstract: Objective To study the role of ubiquitin-conjugating enzyme 9 ( UBC9) in the pyroptosis of homocysteine-induced macrophages mediated by small ubiquitin-like modifier (SUMO) modification. Methods First, the effects of homocysteine at different concentrations (0 μmol / L, 50 μmol / L, 100 μmol / L, 150 μmol / L and 200 μmol / L) on the viability and pyrodeath of mouse macrophages (RAW264. 7) were detected by CCK-8 and Western blot. Western blot was used to detect the expression levels of UBC9, SUMO-1, and the inflammatory cytokine IL-1β in different groups of cells. qRT-PCR was used to detect the mRNA expression of UBC9 before and after RNA interference and the expression of UBC9, pyrogen-related protein, and SUMO-1 after RNA interference. Results After stimulation with 100 μmol / L homocysteine, the effect of macrophage activity was minimal, and NLRP3 and Caspase-1 were the proteins with the most obvious increase in expression (P<0. 05). Compared with the Control group, the Hcy group’ s expression of IL-1β and SUMO-1 was increased (P<0. 01). Compared with the Control group, the Hcy group’ s UBC9 protein and mRNA levels were increased (P<0. 05). The expression of NLRP3, Caspase-1, IL-1β, UBC9, and SUMO-1 was decreased in the siUBC9 + Hcy group compared with the si-NC+Hcy group (P<0. 01). Conclusions Homocysteine induces pyroptosis in macrophages, and its mechanism of action is related to the up-regulation of UBC9 to induce SUMO modification.
2024, 34(9):12-18.DOI: 10.3969/j.issn.1671-7856.2024.09.002
Abstract: Objective To investigate the effect of miR-22-3p on pyroptosis of vascular smooth muscle cells (VSMCs) induced by homocysteine (Hcy). Methods Human VSMCs were cultured in vitro and divided into a Control group (0 μmol/L Hcy) and a Hcy group (100 μmol/L Hcy). After intervention, expression levels of pro Caspase-1,gasdermin D (GSDMD), N-GSDMD, and NLR family pyrin domain containing 3 (NLRP3) were detected by Western blot. MiR-22-3p expression was determined by quantitative real-time reverse-transcription polymerase chain reaction. Interleukin (IL)-1β and IL-18 levels in the supernatant were measured by enzyme-linked immunosorbent assay. Cells were also transfected with control miR-22-3p (miR-22-3p-NC), miR-22-3p-mimic, and miR-22-3p-inhibitor, to observe the effects on VSMC pyroptosis induced by Hcy. Results Expression levels of pro Caspase-1, GSDMD, N-GSDMD, and NLRP3 in VSMCs were increased (P<0.05), IL-1β and IL-18 levels were increased (P<0.01), and the relative expression level of miR-22-3p was reduced (P<0.01) in the Hcy group compared with the Control group. Transfection with miR-22-3p-mimic significantly decreased the expression levels of pro Caspase-1, GSDMD, N-GSDMD, and NLRP3 in VSMCs (P<0.01), and significantly decreased levels of IL-1β and IL-18 (P<0.01), while transfection with miR-22-3p inhibitor significantly increased the expression levels of pro Caspase-1, GSDMD, N-GSDMD, and NLRP3 in VSMCs (P<0.01) and significantly increased the levels of IL-1β and IL-18 (P<0.05). Conclusions MiR-22-3p may delay Hcy induced VSMC pyroptosis.
2024, 34(11):34-42.DOI: 10.3969/j.issn.1671-7856.2024.11.005
Abstract: Objective To study the effect of DNA methyltransferase 1 (DNMT1) on sm-like protein-4 (LSM4) in hepatocyte apoptosis in mice induced with Hcy. Methods 12 ApoE-/- mice were divided into two groups: normal diet (ND, n=6) and high methionine diet (HMD, n=6) groups. Normal hepatocytes of NCTC1469 were divided into a normal group (control, 0 μL/L Hcy), Hcy intervention group (Hcy, 100 μL/L Hcy), NC siRNA-transfected control group (si NC group, 0 μmol/L Hcy), LSM4 siRNA-transfected group (si-LSM4 group, 0 μmol/L Hcy), DNMT1 siRNA transfected group (si-DNMT1 group, 0 μmol/L Hcy), NC siRNA-transfected Hcy intervention group (Hcy+si-NC group, 100 μmol/L Hcy), LSM4 siRNA-transfected Hcy intervention group (Hcy+si-LSM4 group, 100 μmol/L Hcy), and DNMT1 siRNA-transfected Hcy intervention group (Hcy+si-DNMT1 group, 100 μmol/L Hcy). Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect differences in LSM4 protein expression in mouse tissues (HMD and ND) and hepatocytes (control and Hcy). Western blot was used to detect the expression of Bcl2-associated X (Bax) and B-cell lymphoma-2 (Bcl-2). The cell apoptosis rate in the Control, Hcy, Hcy+si-NC, and Hcy+si-LSM4 groups were detected by flow cytometry. MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region. The expression of LSM4 in the Hcy+si-DNMT1 group was detected by qRT-PCR and Western blot. Results The expression of LSM4 in HMD,Hcy group was higher than that in the ND and Control group (P<0.05). Bax protein expression was significantly higher, but Bcl-2 was significantly lower in Hcy group compared with those of the Control group (P<0.05). The expression of Bax protein was significantly lower, but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group (P<0.05). The cell apoptosis rate in the Hcy group was higher than that in the Control group (P<0.05), while the apoptosis rate in the Hcy+si-LSM4 group was lower than that in the Hcy+si-NC group (P<0.05). MethPrimer database analysis showed that the promoter region of LSM4 was GC-rich, and there was one CpG island. Compared with the Hcy + si-NC group, the Hcy+si-DNMT1 group’s expression of LSM4 protein was increased (P<0.05). Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression, thereby promoting Hcy-induced apoptosis of mouse hepatocytes.
2023, 33(5):15-23.DOI: 10. 3969 / j.issn.1671-7856. 2023. 05. 003
Abstract:Objective To establish a rat model of chronic kidney disease with hyperhomocysteinemia by high methionine feeding and 5/6 nephrectomy. Then, to evaluate the model by comparing it with the simple high methionine feeding model and simple nephrectomy model. Methods Five-week-old rats were randomly divided into four groups: sham, high methionine (feeding on a high methionine diet), nephrectomy (5/6 nephrectomy), and compound model (high methionine diet and 5/6 nephrectomy) groups. Arterial blood pressure, echocardiography, and serum biochemical indices were measured. Changes in the tension of isolated thoracic aortic vascular rings were observed and expression of pp85, p-Akt, and p-eNOS (Ser1177) proteins was detected. HE staining was used to observe pathological changes in renal, myocardial, and aortic tissue. Results Compared with the sham group, arterial blood pressure of the three model groups increased continuously (P<0.01). Arterial blood pressure of compound model group was significantly higher than high methionine group and nephreetomy group(P<0.05). Compared with the sham group, echocardiography indexes LVEDd, LVEDV, LVESV, and SV of the three model groups were significantly different (P<0.05). Compared with the sham group, serum homocysteine and methionine in high methionine and compound model groups were increased (P<0. 01), while, creatinine, urea nitrogen, total cholesterol, and high-density lipoprotein in serum were increased significantly (P<0. 05) and triglyceride was significantly decreased (P<0. 05) in nephrectomy and compound model groups. Compared with the sham group, vascular ring relaxation curves of model groups induced by vasodilators shifted to the right, and the Emax and EC50 of the relaxation effect induced by acetylcholine and sodium nitroprusside in the compound model group were significantly different from those in the simple model group (P<0. 05). Compared with the sham group, expression of p-Akt and p-eNOS in aortic tissue of the compound model group was statistically significant (P<0. 05). Pathological changes were observed in renal, myocardial, and aortic tissues of the model animals, among which the compound model group had more severe pathological changes. Conclusions A rat model of chronic kidney disease with hyperhomocysteinemia can be established by combining a high methionine diet and 5/6 nephrectomy. Compared with the simple models, the functional and morphological changes are significantly different in the compound model, which can be applied to fundamental research on chronic kidney disease with hyperhomocysteinemia.
2022, 32(9):1-9.DOI: 10. 3969 / j.issn.1671-7856. 2022. 09. 001
Abstract: Objective To investigate the role of the microRNA miR-488-3p in homocysteine-mediated hepatocyte autophagy through targeted regulation of the RAS oncogene family member RAP1A. Methods Human normal hepatocytes(HL-7702) were routinely cultured in vitro and administered 100 μmol / L Hcy for 24 h. Western blot was used to detect expression levels of autophagy-related proteins LC3, p62 and RAP1A in hepatocytes, whereas qRT-PCR was used to detect the expression of miR-488-3p and RAP1A in these cells. Following transfection of miR-488-3p inhibitor and mimics, qRTPCR and western blot were used to detect the transfection efficiency of miR-488-3p and its effect on LC3 and p62 protein expression. TargetScan was used to predict the correlation between miR-488-3p and RAP1A genes, whereas western blotting was used to detect changes in the expression of RAP1A, a potential downstream target protein of miR-488-3p. Pearson’s correlation coefficient was used to evaluate potential correlations between expression levels of miR-488-3p and autophagy-related proteins in hepatocytes. Results Compared with the control group, expression levels of autophagyrelated proteins LC3, RAP1A and miR-488-3p in the Hcy group were increased (P<0. 01), whereas expression of p62 was significantly decreased (P<0. 01). Following administration of miR-488-3p inhibitor and mimics, expression levels of LC3 and RAP1A proteins in the miR-488-3p inhibitor group were significantly decreased (P<0. 01) and expression of p62 was significantly increased (P<0. 01) compared with the NC-inhibitor group. Compared with the NC-mimics group, expression levels of LC3 and RAP1A proteins in the miR-488-3p mimics group were significantly increased ( P < 0. 01 ), and expression of p62 was significantly decreased (P<0. 01). Further mechanistic studies showed that RAP1A is a downstream target gene of miR-488-3p that is positively regulated by this miRNA. Pearson’ s correlation analysis indicated that the expression level of miR-488-3p was positively correlated with protein expression of LC3 ( r= 0. 9329, P= 0. 0002), but negatively correlated with p62 protein expression ( r= - 0. 8086, P= 0. 0083). Conclusions miR-488-3p is highly expressed in Hcy-mediated hepatocytes and can promote their autophagy by targeting expression of RAP1A.
2014, 24(4):43-46.DOI: 10.3969.j.issn.1671.7856.2014.004.010
Abstract:Objective To explore the influence of carotid artery balloon injury on the formation of atherosclerotic lesions induced by hyperhomocysteinemia in rabbits. Methods Twenty New Zealand rabbits were divided into control group and model group (n=10) randomly. The left carotid arteries were injured by balloon catheterization in all rabbits. After operation, the model group was given methionine 80 mg/kg per day by subcutaneous injection for 24 weeks to induce atherosclerotic lesions, and the control group was given the same amount of normal saline. All rabbits were killed at the 24th week. ELISA was used to detect the content of homocysteine and HE staining was used to observe the pathological changes in the arterial wall. The arterial wall thickness was measured using an image analysis software. Results After 24 weeks, the level of serum homocysteine of the model group was significantly increased and pathological changes of aortic wall were observed in different degrees compared with that of the control group (P<0.05). But the carotid artery wall thickness of model group after balloon injury had no visible changes compared with that of the control group. Conclusion Balloon injury of carotid artery has no significant effect on the formation of atherosclerotic lesions induced by hyperhomocysteinemia.