植物TIR1/AFBs基因家族研究进展

TIR1/AFBs基因家族是一种存在于细胞核中的生长素受体,属于F-box蛋白基因中的一个小亚族。它们通过与相关生长素相结合活化转录因子来促进基因的表达,从而进行调控,是生长素信号转导过程中的关键部分。为了深入研究生长素信号转导机制,从TIR1/AFBs基因家族的发现与结构,家族成员表达模式的差异及对植物生长发育方面的调节等方面概括介绍了TIR1/AFBs基因家族的分子调控机制,总结了TIR1/AFBs基因的功能。最后探讨了TIR1/AFBs基因的研究方向。     

蛋鸡育成期和产蛋高峰期生殖激素水平及激素受体基因表达规律研究

【目的】探究蛋鸡育成期和产蛋高峰期相关生殖激素含量及其受体基因mRNA表达量在24 h内的变化规律与差异,分析血液生殖激素含量变化与产蛋排卵行为的关系。【方法】以育成期和产蛋高峰期的罗曼蛋鸡为研究对象,监测其在产蛋高峰期的产蛋时间点,运用酶联免疫吸附法(ELISA)测定2个时期蛋鸡血液中前列腺素(PGs)、雌二醇(E2)、促卵泡素(FSH)、黄体生成素(LH)含量在24 h内的动态变化,实时荧光定量PCR技术检测相应时间点的前列腺素F受体(PTGFR)、雌激素受体(ERα)、促卵泡素受体(FSHR)和黄体生成素受体(LHR)基因mRNA在输卵管子宫部的日表达水平。【结果】蛋鸡产蛋时间集中在早上08:00-10:00。产蛋高峰期时,一天中06:00、10:00、14:00、18:00的PGs含量显著高于育成期(P0.05),E2含量在06:00和18:00极显著高于育成期(P0.01),FSH含量在18:00显著高于育成期(P0.05),LH含量在14:00、22:00和02:00显著高于育成期(P0.05)。育成期除LH外,其余3种激素含量在一天中波动不大。产蛋高峰期PGs含量在产蛋前显著高于产蛋后,PTGFR mRNA表达量在产蛋前后4 h均显著(P0.05)升高;FSH和E2含量在产蛋后(18:00)极显著(P0.01)上升。子宫组织的激素受体基因表达量变化与相应血液激素含量变化一致。【结论】蛋鸡产蛋时间集中于上午,产蛋高峰期各生殖激素含量总体高于育成期,蛋鸡育成期和产蛋高峰期的血液生殖激素水平变化规律与输卵管子宫部激素受体基因mRNA表达量的变化规律基本一致。PGs参与蛋鸡产蛋高峰期的产蛋和排卵过程,E2和FSH可能在蛋壳形成过程中发挥调控作用。     

Protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats

AIM: To investigate the protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats.METHODS: SPF male Sprague-Dawley rats (n=40) were randomly divided into 5 groups: normal control (NC) group, high fat (HF) group, high fat+L6H4 treatment (FT) group, diabetes mellitus (DM) group and DM+L6H4 treatment (DT) group. The rats in the later 4 groups were fed with high-fat diet. After 4 weeks of high-fat diet fee-ding, the rats in DM and DT groups were intraperitoneally injected with streptozotocin to induce type 2 diabetes melliutus. The rats in FT and DT groups were given L6H4 by gavage for 8 weeks. Blood glucose and blood lipid levels were detected biochemically. Fasting serum insulin (FINS) level was measured by radioimmunoassay and insulin resistance index (HOMA-IR) was calculated. Serum adiponectin (APN) level was measured by ELISA. The morphological changes of the diaphragm were observed under light and transmission electron microscopes. Lipid deposition and the activity of succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) were observed by enzyme histochemical staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the diaphragm were measured by thiobarbituric acid method and hydroxylamine method, respectively. The protein expression of adiponectin receptor 1 (AdipoR1) in the diaphragm was determined by immunohistochemistry and Western blot. RESULTS: The levels of blood lipids, blood glucose, FINS and HOMA-IR in HF and DM groups were higher than those in NC group, but decreased after L6H4 treatment. The serum APN level in HF and DM groups was lower than that in NC group, but increased after treatment with L6H4. The muscle fibers of the diaphragm were shrunk, fat particles accumulated in the muscle fibers, and the mitochondria were slightly swollen in HF and DM groups. The diaphragmatic fibrosis was obvious in DM group. These lesions were relieved after L6H4 treatment. Compared with NC group, the level of MDA and the activity of SDH and NADH-TR in the diaphragm were increased in HF and DM groups, but decreased after treatment with L6H4. The activity of SOD and the expression of AdipoR1 in the diaphragm were lower than those in NC group, but increased after L6H4 treatment.CONCLUSION: The curcumin analogue L6H4 exerts a protective effect on diaphragm in type 2 diabetic rats. The strengthened protein expression of AdipoR1, the increased serum level of APN, and anti-lipid peroxidation may be involved in the process.     

Albumin overload aggravates kidney injury and renoprotective effect of simvastatin in adriamycin nephropathy mice

AIM: To investigate the aggravating effect of albumin overload on the kidney injury induced by lipid nephrotoxicity, and to observe the renoprotective effect of simvastatin (SIMV) on adriamycin nephropathy (ADR) mice.METHODS: SPF healthy male BALB/c mice were randomly divided into control group, ADR group, ADR with bovine serum albumin (BSA) overload (ADR+BSA) group, and ADR with both BSA overload and SIMV treatment (ADR+BSA+SIMV) group. All mice were uninephrectomized under general anesthesia 2 weeks before setting up ADR model. ADR+BSA model started to be set up 4 weeks later. At the end of the 0th, 2nd, 6th, 10th and 14th weeks, 24 h urinary protein was evaluated. At the end of the 14th week, the serum biochemical indexes and the kidney pathological changes were observed, and glomerulosclerotic index (GSI) was also evaluated. The cholesterol in the kidney was measured by enzymic colorimetric method and oil red O staining. The expression of IL-1β, TGF-β1 and low-density lipoprotein receptor (LDLr) in the kidney tissues was determined by real-time PCR. The expression of IL-1β and IL-17 was measured by immunohistochemistry and the expression of IL-17 in the kidney was measured by ELISA.RESULTS: Compared with control group, the expression of IL-1β, TGF-β1, IL-17 and LDLr, and cholesterol content in the kidney and the GSI were all significantly increased in ADR group (P<0.05). Compared with ADR group, 24 h urinary protein, serum creatinine, the expression of IL-1β, IL-17 and LDLr, and cholesterol content in the kidney were all significantly increased in ADR+BSA group (P<0.05). Treatment with SIMV significantly decreased the expression of IL-1β, TGF-β1, IL-17 and LDLr. The accumulation of cholesterol in the kidney and the GSI were also decreased (all P<0.05).CONCLUSION: Inflammation aggravates the lipid deposition and glomerular sclerosis by increasing the expression of LDLr in ADR mice. Albumin overload further accelerates the progressive kidney damage by regulating the expression of IL-1β, TGF-β1 and IL-17, which promotes the increase in LDLr. The beneficial effect of SIMV might be mediated by its anti-inflammatory effect.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Screening of lentiviral vectors carrying effective siRNA targeting S1P2 gene

AIM:To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS:SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups:SHR siRNA-1,SHR siRNA-2,SHR siRNA-3,SHR GFP,SHR control (SHR non-transfection group),and SD control (SD rat control group).Each group had 5 samples with 1.0×10⁵ cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells,the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2,ROCK1,ROCK2 and eNOS in the corpus cavernosum smooth muscle cells,and the mRNA expression of S1P2,ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS:The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group,the mRNA levels and the protein expression of S1P2,ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes,while those in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group,but that in SD control group was significantly higher than that in SHR control group.CONCLUSION:Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR,and by silencing the S1P2 expression,the expression of ROCK1 and ROCK2 is inhibited.Among them,siRNA-1 has the highest inhibitory efficiency.     

Effect of eleutheroside B/E on proliferation and expression of PPARγ and TGF-β1 in HBZY-1 cells cultured with high glucose

AIM: To explore the effects and mechanism of eleutheroside (ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose. METHODS: The HBZY-1 cells were cultured under high glucose condition. The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth regulation curve of the cells. The cells were divided into 6 groups: low glucose (LG) group, high glucose (HG) group, high glucose plus ETS-B/E (low dose, medium dose and high dose) groups, and high glucose plus losartan (LTG) group. After all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγ was detected by immunocytochemistry and Western blotting. RESULTS: The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation. At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells (P<0.05). The expression of TGF-β1 was significantly inhibited, and the expression of PPARγ was significantly promoted by ETS-B/E (P<0.05). ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration- and time-dependent manner. CONCLUSION: ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγ expression.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.