miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury

ObjectiveThis study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2).MethodsSerum levels of miR-34b-5p, TNF-α, IL-1β, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1β, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2.ResultsThe expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells.ConclusionmiR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.     

LncRNA MALAT1-deficiency restrains lipopolysaccharide (LPS)-induced pyroptotic cell death and inflammation in HK-2 cells by releasing microRNA-135b-5p

Long non-coding RNAs (LncRNAs) participate in the regulation of chronic kidney disease (CKD), and acute kidney injury (AKI) is identified as an important risk factor for CKD. This study investigated the involvement of a novel LncRNA MALAT1 in regulating lipopolysaccharide (LPS)-induced cell pyroptosis and inflammation in the human renal tubular epithelial HK-2 cells. Here, the HK-2 cells were subjected to LPS (2 μg/mL) treatment to establish cellular AKI models in vitro, and we validated that LPS triggered NLRP3-mediated pyroptotic cell death, promoted cell apoptosis and inflammation-associated cytokines secretion to induce HK-2 cell injury. Then, a novel LncRNA MALAT1/miRNA (miRNA)-135b-5p axis was verified to rescue cell viability in LPS treated HK-2 cells by targeting NLRP3. Mechanistically, miRNA-135b-5p bound to LncRNA MALAT1, and LncRNA MALAT1 positively regulated NLRP3 through acting as RNA sponger for miRNA-135b-5p. Further gain- and loss-of-function experiments evidenced that both LncRNA MALAT1 ablation and miRNA-135b-5p overexpression reversed LPS-induced cell pyroptosis, apoptosis, and inflammation in the HK-2 cells, and the protective effects of LncRNA MALAT1 knock-down on LPS-treated HK-2 cells were abrogated by silencing miRNA-135b-5p. In general, our study firstly investigated the role of the LncRNA MALAT1/ miRNA-135b-5p/NLRP3 signaling cascade in regulating LPS-induced inflammatory death in HK-2 cells.     

LncRNA MALAT1 Promotes Lung Cancer Proliferation and Gefitinib Resistance by Acting as a miR-200a Sponge

IntroductionLung cancer is a major public health problem, as the second causes of cancer related death worldwide, with relatively low survival rates, and accessible drug resistance. Long non-coding RNAs (LncRNAs) have been identified as activator in lung cancer with elusive mechanisms. We aimed to detect the regulation of LncRNA MALAT1 in the proliferation and gefitinib resistance in lung cancer cells.MethodsMALAT1 in A549 and HCC 1299 human lung adenocarcinoma cell lines was silenced by shRNA or overexpressed using plasmid, and the cell viability and cell proliferation were evaluated by MTT assay and soft agar colony formation assay. RNA levels were detected by RT-PCR, and the protein expression was measured by western blot. The binding between MALAT1 and miR-200a was validated by luciferase reporter assays using pSi-Chech 2 vectors.ResultsThe cell viability and proliferation of A549 cells transfected with MALAT1 shRNA were significantly lower than the control. The MALAT1 expression in gefitinib resistant A549 cells was upregulated. miR-200a significantly inhibited the fluorescence of pSi-Check 2 vector with MALAT1 gene, suggesting the direct binding between MALAT1 and miR-200a. In addition, LncRNA MALAT1 promotes ZEB1 expression in A549 cells.ConclusionOur study showed that MALAT1 promoted the proliferation and gefitinib resistance of lung cancer cells by sponging miR-200a, which regulates expression of ZEB1 in the A549 cells. This MALAT1/miR-200a axis could serve as new therapeutic target for lung cancer treatment.     

Downregulation of miR-574-5p inhibits HK-2 cell viability and predicts the onset of acute kidney injury in sepsis patients

BackgroundIncreased levels of microRNA-574-5p (miR-574-5p) have been found to be associated with increased survival of septic patients, indicating the potential role of miR-574-5p in protecting against septic progression and complications. Acute kidney injury (AKI) is one of the most common and serious complications of sepsis. Therefore, the aim of this study was to test these hypotheses: (1) in a renal cell culture line (HK-2), upregulated expression of miR-574-5p increases, and downregulated expression of miR-574-5p decreases cell viability, and (2) serum levels of miR-574-5p from patients with sepsis and AKI are lower than those of patients with sepsis but no AKI.MethodsThe expression of miR-574-5p was regulated by cell transfection in HK-2 cells, and HK-2 cell viability was measured using the Cell Counting Kit-8. Serum miR-574-5p expression was analyzed using qRT-PCR. The predictive value of miR-574-5p for AKI onset was evaluated using the receiver operating characteristic curve and logistic regression analysis.ResultsThe overexpression of miR-574-5p promoted HK-2 cell viability. Fifty-eight sepsis patients developed AKI, who had significantly lower miR-574-5p expression. miR-574-5p expression was decreased with AKI stage increase and correlated with kidney injury biomarker and had relatively high accuracy to predict AKI occurrence from sepsis patients.ConclusionOverexpression of miR-574-5p in cultured HK-2 cells increases cell viability and knocked-down expression of miR-574-5p decreases cell viability. Consistently, septic patients with AKI were found to have less upregulation of miR-574-5p expression compared to septic patients without AKI. Thus, serum miR-574-5p may provide a novel biomarker for septic AKI.     

长链非编码核糖核酸LINC00261通过miR-148b-3p/PTEN途径对高糖环境中HK-2细胞的保护作用

目的探讨长链非编码RNA(lncRNA)LINC00261在糖尿病肾病中的表达,及其可能通过微小核糖核酸miR-148b-3p/PTEN途径对高糖环境中HK-2细胞的保护作用。 方法选择2016年3月至2018年5月本院的19例糖尿病肾病患者和23例健康对照者,采集血样,通过实时定量PCR(qRT-PCR)测定LINC00261和miR-148b-3p的表达。在细胞实验中,将HK-2肾小管上皮细胞分为7组：正常葡萄糖组(5.5 mmol/L培养,NG组)、高葡萄糖糖组(30.0 mmol/L培养,HG组)、其余5组也均为高葡萄糖培养：空质粒转染pcDNA(HG+pcDNA组)、转染LINC00261(HG+pcDNA-LINC00261组)、转染阴性对照anti-miR-NC(HG+anti-miR-NC组)、转染抑制剂anti-miR-148b-3p(HG+anti-miR-148b-3p组)、LncRNA和miRNA同时转染(HG+pcDNA-LINC0026+miR-148b-3p组)。应用Western印迹、细胞计数试剂盒8和流式细胞术分别检测PTEN蛋白表达、细胞增殖与凋亡。测超氧化物歧化酶(SOD)试剂盒和丙二醛(MDA)试剂盒分别检测SOD活性和MDA含量。双荧光素酶报告实验用于LINC00261、miR-148b-3p、PTEN的相互关系鉴定。 结果与健康对照者比较,糖尿病肾病患者的LINC00261表达明显降低,而miR-148b-3p表达则明显增高(P<0.05)。过表达LINC00261或抑制miR-148b-3p后,高糖环境中HK-2细胞的miR-148b-3p表达、细胞凋亡及MDA含量均下降,而PTEN蛋白表达、细胞增殖及SOD活性均增高(P<0.05)。 结论LINC00261可能通过调控miR-148b-3p/PTEN途径,促进高糖环境中HK-2肾小管上皮细胞增殖、降低氧化应激、减少细胞凋亡。     

p53 inhibition attenuates cisplatin-induced acute kidney injury through microRNA-142-5p regulating SIRT7/NF-κB

Renal tubular epithelial cell apoptosis is the main mechanism of cisplatin-induced acute kidney injury. The role of microRNAs (miRNAs) in the apoptosis of renal tubular epithelial cells has been suggested, but the underlying mechanism has not been fully elucidated. We used microarray analysis to identify miR-142-5p involved in cisplatin-induced acute kidney injury. miR-142-5p was down-regulated in human renal tubular epithelial (HK-2) cells with cisplatin treatment. Notably, the overexpression of miR-142-5p attenuated the cisplatin-induced HK-2 cell apoptosis and inhibition of miR-142-5p aggravated cisplatin-induced HK-2 cell apoptosis. During cisplatin treatment, p53 was activated. The inhibition of p53 by pifithrin-α attenuated the cisplatin-induced kidney injury and up-regulated miR-142-5p expression. We also identified the Sirtuin7 (SIRT7) as a target of miR-142-5p. Furthermore, we demonstrated that the inhibition of SIRT7 prevented cisplatin-induced HK-2 cell apoptosis and decreased the expression of nuclear factor kappa B (NF-κB). Our data revealed that p53 inhibition could attenuate cisplatin-induced acute kidney injury by up-regulating miR-142-5p to repress SIRT7/NF-κB. These findings may provide a novel therapeutic target of cisplatin-induced acute kidney injury.     

Effect of cyclosporine A on autophagy-lysosomal pathway in tubular epithelial cells

Objective To investigate the effect of cyclosporine A (CsA) on autophagy-lysosomal pathway in tubular epithelial cells. Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3, 5 and 10 μmol/L) of CsA for 24 h. Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis, respectively. Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay. Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (tfLC3) plasmid by laser confocal microscope. The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis. Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P＜0.01), but the number of apoptotic cells was markedly increased by CsA treatment (P＜0.05). Compared with the control group, different doses of CsA dramatically increased the expressions of LC3-Ⅱ(P＜0.01) and p62 (P＜0.05) in HK-2 cells. Moreover, HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes. In HK-2 cells, exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P＜0.01). Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.     

Mechanism of miR-365 in regulating BDNF-TrkB signal axis of HFD/STZ induced diabetic nephropathy fibrosis and renal function

Purpose

Diabetic nephropathy (DN) is one of the most serious complications of diabetes that leads to decline of renal function. Although numerous studies have revealed that microRNAs (miRNAs) play essential roles in the progression of DN, whether miR-365 is involved remains elusive.

Methods

The successful construction of DN model was confirmed by ELSIA, hematoxylin–eosin (HE) and Masson staining assay. The expression of miR-365 was detected through RT-qPCR. The levels of BDNF, p-TrkB, α-smooth muscle actin (SMA), collagen IV (Col.IV), transforming growth factor-β1 (TGF-β1), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were evaluated by western blot, IF or ELISA assays. Luciferase reporter assay was used to detect the interaction between miR-365 and BDNF.

Results

The DN mice model was induced by streptozotocin (STZ). Then miR-365 expression was found to upregulate in tissues of DN rat. Furthermore, elevated expression of miR-365 was found in high glucose (HG)-treated HK-2 cells. Silencing of miR-365 suppressed the accumulation of ECM components and secretion of inflammatory cytokines in HK-2 cells. In addition, it was demonstrated that miR-365 could target BDNF. The protein levels of BDNF and p-TrkB were negatively regulated by miR-365 in HK-2 cells. Moreover, inhibition of miR-365 suppressed the levels of SMA, Col.IV, TGF-β1, TNF-α, and IL-6, indicating the renal fibrosis was inhibited by miR-365 knockdown.

Conclusion

MiR-365 could regulate BDNF-TrkB signal axis in STZ induced DN fibrosis and renal function. The results of the current study might provide a promising biomarker for the treatment of DN in the future.

     

Neutrophil gelatinase-associated lipocalin worsens ischemia/reperfusion damage of kidney cells by autophagy

This study aimed to explore the influence of neutrophil gelatinase-associated lipocalin on autophagy and its role in ischemia/reperfusion injury in human kidney-2 (HK-2) cells during acute kidney injury (AKI). HK-2 cells were given hypoxia/reoxygenation treatment for different times to simulate ischemia/reperfusion injury. Autophagy was evaluated by western blot and immunofluorescence of GFP-LC3. Cell viability was tested to reflect the degree of cell damage. The autophagy inhibitor 3-MA was used to inhibit autophagy and determine the role of autophagy in ischemia/reperfusion injury. HK-2 cells were hypoxia for 1?h, followed by reoxygenation treatment for 24?h. These cells were then exposed to human recombinant protein neutrophil gelatinase-associated lipocalin (NGAL) (50, 100, 200, 400, or 1000?ng/mL) with or without 3-MA. Our results showed that autophagy was induced by hypoxia treatment and was further enhanced by reoxygenation after hypoxia treatment. Cell viability was decreased with the inhibition of autophagy in the process. Autophagic flux was further induced with NGAL (>200?ng/mL), while cell viability declined in this condition. Cell viability was recovered when autophagy was inhibited. These results indicate that autophagy plays, in part, a protective role in renal ischemia/reperfusion injury. Furthermore, the data suggest that NGAL strengthens the level of autophagy in this process. Overall, a large quantity of NGAL produced by renal proximal tubular epithelial cells may induce excessive autophagy and increase renal ischemia/reperfusion injury in acute kidney injury.     

Inhibition of miR-495-3p ameliorated sevoflurane induced damage through BDNF/ERK/CREB signaling pathways in HT22 cells

ObjectiveSevoflurane is used in anesthesia for surgery including in organ transplantation. We investigated the role of a non-coding single-stranded microRNA, miR-495-3p, in the sevoflurane-induced neurotoxicity using a mouse hippocampal neuronal cell line (HT22).MethodsThe levels of miR-495-3p in sevoflurane-exposed mice and HT22 cells were determined via RT-qPCR. The role of miR-495-3p on cell viability and apoptosis were determined by CCK-8 and flow cytometric assay, respectively. Western blotting was explored to measure levels of Bax, Bcl-2, active caspase 3, BDNF, p-ERK/ERK and p-CREB/CREB in HT22 cells. ELISA assay was used to examine the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in cells. Dual luciferase reporter assay was used to explore the interaction of miR-495-3p and BDNF.ResultsThe level of miR-495-3p was increased sevoflurane-exposed mice and in sevoflurane-treated HT22 cells. Downregulation of miR-495-3p inhibited sevoflurane-induced apoptosis and promoted cell proliferation by upregulating the proteins of Bcl-2 and downregulating the expressions of Bax and active caspase-3 in HT22 cells. In addition, inhibition of miR-495-3p alleviated sevoflurane-induced oxidative injuries in HT22 cells via decline of ROS and upregulation of SOD and GPX. MiR-495-3p can inhibit the ERK/CREB pathway by targeting BDNF.ConclusionDownregulation of miR-495-3p can decrease oxidative status in HT22 cells and alleviate sevoflurane-induced cytotoxicity through stimulating the BDNF/ERK/CREB pathway.     

MicroRNA-503 regulates high-glucose induced apoptosis of renal tubular epithelial cells by targeting Bcl-2

Objective To investigate the expression vibration of microRNA-503(miR-503) and its effect on target gene Bcl-2, caspase enzyme activity and apoptosis of human renal tubular epithelial cells (HK-2) induced by high glucose, and to clarify the pathogenesis of renal tubular injury induced by high glucose. Methods HK-2 cells were cultured in normal glucose group (NG), mannitol hypertonic control group (MA), and high glucose group (HG). The morphology of apoptotic cells was observed using inverted microscope. The expression of miR-503 was determined using real-time quantitative PCR. The apoptosis rate of HK-2 cells was detected by Annexin Ⅴ-FITC double dye using flow cytometry instrument. The expression of Bcl-2 and cleaved caspase-9 were detected by Western blotting. Results In the high glucose and mannitol groups HK-2 cell, an obviously increased apoptotic rate was observed under inverted microscope compared with normal glucose group (P＜0.05). MA and HG up-regulated miR-503 expression (P＜0.01), down-regulated anti-apoptotic protein Bcl-2 expression (P＜0.05) and up-regulated cleaved caspase-9 (P＜0.05). Conclusions The expression of miR-503 increases in HK-2 cells cultured by high glucose and mannitol. MiR-503 promotes apoptosis of HK-2 cells via activating mitochondrial apoptotic pathways and enhancing cleaved caspase-9 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. The miR-503 may be involved in diabetic tubular injury and may be a new therapeutic target of DN.     

CircHIPK3 alleviates inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis in spinal cord injury

BackgroundSpinal cord injury (SCI) is one of the serious neurological diseases with high morbidity which may be treated with hematopoietic stem cell (HSC) transplants. Circular RNAs (circRNAs) play vital roles in SCI. The study aimed to reveal the function and mechanism of circRNA homeodomain interacting protein kinase 3 (HIPK3) in SCI.MethodsSCI model in vitro was established by treating neuronal cells AGE1.HN with oxygen-glucose deprivation (OGD) and CoCl2. The levels of circHIPK3, miR-382-5p and dual specificity phosphatase 1 (DUSP1) were examined using quantitative real-time PCR (qRT-PCR) or western blot assay. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors (IL-6 and TNF-α). Cell proliferation and apoptosis were evaluated by 5′-ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry. Caspase-3 Colorimetric Assay Kit was used to detect aaspase-3 activity. The interactions among circHIPK3, miR-382-5p and DUSP1 were confirmed by dual-luciferase reporter and RNA immunoprecipitation assays.ResultsCircHIPK3 and DUSP1 were down-regulated, while miR-382-5p was up-regulated in OGD-induced AGE1.HN cells. Overexpression of circHIPK3 suppressed inflammatory response and cell apoptosis and promoted proliferation in OGD-induced AGE1.HN cells by sponging miR-382-5p. CircHIPK3 regulated DUSP1 expression by targeting miR-382-5p. MiR-382-5p inhibition hindered inflammatory response of IL-6 and TNF-α and neuronal apoptosis and promoted apoptosis via targeting DUSP1.ConclusionCircHIPK3 overexpression alleviated OGD-induced AGE1.HN cell inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis, indicating that circHIPK3 might be a promising therapeutic target for SCI.     

High-glucose induced toxicity in HK-2 cells can be alleviated by inhibition of miRNA-320c

Diabetic nephropathy (DN) is a major healthcare challenge worldwide. MiRNAs exert a regulatory effect on the progress of DN. Our study proposed to investigate the miR-320c expression and its function on the pathogenesis of DN in vitro. The level of miR-320c in HK-2 cells was quantified by RT-qPCR. Cell morphology, invasion, and migration were observed by optical microscope, Transwell invasion assay, and scratch wound assay. Then, the levels of PTEN, α-SMA, vimentin, E-cadherin, p-PI3K, PI3K, AKT, and p-AKT were analyzed through western blotting. A Dual-luciferase reporter assay was conducted to explore the target relationship between miR-320c and PTEN. It was discovered that miR-320c was over-expressed in high glucose (HG)-treated HK-2 cells. Furthermore, inhibition of miR-320c could alleviate the epithelial-mesenchymal transition (EMT) of HG-induced HK-2 cells and retain the normal morphology of HK-2 cells. Additionally, the miR-320c inhibitor decreased the invasiveness and migration of HG-treated HK-2 cells. Next, the target gene of miR-320c, PTEN, was identified, and the function of miR-320c was reversed by down-regulation of PTEN. Finally, we found inhibition of miR-320c restrained the PI3K/AKT pathway. Therefore, inhibition of miR-320c could alleviate toxicity of HK-2 cells induced by HG via targeting PTEN and restraining the PI3K/AKT pathway, illustrating that miR-320c may act as a new biomarker in the diagnosis of DN.     

Knockdown of circ_0026579 ameliorates lipopolysaccharide (bacterial origin)-induced inflammatory injury in bronchial epithelium cells by targeting miR-338-3p/TBL1XR1 axis

BackgroundCircular RNAs (circRNAs) play an important regulatory role in human diseases including organ allograft rejection. The aim of this study is to clarify the functional role and molecular mechanism of circ_0026579 RNA in lipopolysaccharide (LPS)-induced bronchopneumonia injury.Materials and methodsBronchial epithelial BEAS-2B cells were treated with LPS to mimic an in vitro model for bronchopneumonia. Cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Flow cytometry assay was used to assess cell apoptosis. Caspase-3 activity was analyzed by Caspase-3 activity assay kit. The expression levels of circ_0026579 RNA, miR-338-3p, and transducin β-like 1× related protein 1 (TBL1XR1) RNA were determined by RT-qPCR. The protein level was quantified by western blot assay. The correlation between miR-338-3p and circ_0026579 or TBL1XR1 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays.ResultsLPS treatment repressed proliferation but induced apoptosis and inflammatory response in BEAS-2B cells. Circ_0026579 RNA was highly expressed in patients with pneumonia. Besides, the expression levels of circ_0026579 RNA and TBL1XR1 RNA/protein were upregulated, while miR-338-3p level was decreased in LPS-treated BEAS-2B cells. Knockdown of circ_0026579 RNA or TBL1XR1 protein could abolish LPS-induced cell injury in BEAS-2B cells. Furthermore, we found that circ_0026579 RNA functioned as a “sponge” for miR-338-3p to regulate TBL1XR1 expression. Additionally, silencing circ_0026579 RNA protected BEAS-2B cells from LPS-induced bronchopneumonia injury by regulating TBL1XR1 expression.ConclusionCirc_0026579 RNA knockdown promoted cell proliferation but inhibited apoptosis and inflammation in LPS-induced BEAS-2B cells through regulating miR-338-3p RNA/TBL1XR1 protein axis.     

乙型肝炎病毒X基因对肾小管上皮细胞间质转分化的影响

目的 探讨乙型肝炎病毒(HBV)X基因表达蛋白HBX对体外培养的人近端肾小管上皮细胞株(HK-2)细胞形态及转分化的影响.方法 用分子克隆的方法构建pcDNA3.1-myc-HBX质粒,采用脂质体转染法瞬时转染HK-2细胞,Q-PCR及Western印迹法验证HBX在HK-2细胞中的表达.以未转染质粒和转染空载质粒pcDNA3.1-myc作为对照.用显微镜观察转染pcDNA3.1-myc-HBX质粒后HK-2细胞形态,用Western印迹及Q-PCR法检测细胞转分化标志蛋白α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白(E-cadherin)的表达;ELISA检测细胞上清液中白细胞介素(IL)-1、IL-6和肿瘤坏死因子α(TNF-α)的表达.结果 转染HBX后的HK-2细胞中存在HBX的高表达,证实转染成功.转染pcDNA3.1-myc-HBX质粒的HK-2细胞数量明显下降,细胞形态不规则,细胞状态受损;转染pcDNA3.1-myc-HBX质粒的HK-2细胞可上调E-cadherin及α-SMA表达;细胞上清液中高表达IL-1、IL-6和TNF-α(P＜ 0.01).结论 HBX质粒转染HK-2细胞后可引起细胞数目和形态的变化,并促进肾小管上皮细胞发生上皮间质转分化,肾小管上皮细胞周围炎性微环境的改变可能是其发生上皮间质转分化的原因之一.     

Effect of cysteine-rich protein 61 on oxidative stress in human kidney tubular epithelial cell line after anoxia

Objective To investigate the effect and mechanism of cysteine-rich protein 61(Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia. Methods Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups：control group, Cyr61 group, MAPK inhibitor group (Cyr61+PD98059), p38 inhibitor group (Cyr61+SB203580) and PI3K inhibitor group (Cyr61+Wortmannin). Each group was pretreated for 12 h and then injured by anoxia．The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry. The cellular ROS level was measured by spectro-fluorometry. The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test. The expression of Nrf2 in HK-2 cells was detected by Western blotting. Results Anoxia enhanced the expression of ROS and Nrf2, decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P＜0.05). Cyr61 increased the expression of p-Akt, Nrf2, SOD and CAT in HK-2, and decreased the expression of ROS, at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P＜0.05). Wortmannin inhibited the expression of p-Akt,Nrf2, SOD and CAT, meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P＜0.05). PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P＞0.05). Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2, which enhances the expression of SOD and CAT, and decreases the expression of ROS. Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.