ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway, thus promoting the apoptosis of cardiomyocytes.     

Effect of Linc00152 targeting miR-376c-3p on viability,apoptosis and radiosensitivity of cervical cancer cells

AIM: To investigate the effect of Linc00152 on the viability, apoptosis and radiosensitivity of cervical cancer cells. METHODS: RT-qPCR was used to detect the expression levels of Linc00152 and microRNA-376c-3p(miR-376c-3p) in human cervical cancer HeLa cells and SiHa cells, and normal cervical Ect1/E6E7 cells. The cervical cancer HeLa cells with low Linc00152 expression or miR-376c-3p over-expression were established. MTT assay, flow cytometry, colony formation assay and Western blot were used to determine the cell viability, apoptosis, radiosensitivity and related protein expression. The dual-luciferase reporter assay was used to verify the regulatory relationship between Linc00152 and miR-376c-3p in the HeLa cells. RESULTS: Compared with the Ect1/E6E7 cells, Linc00152 was up-regulated in the HeLa cells and SiHa cells, and miR-376c-3p was down-regulated (P < 0.05). Low expression of Linc00152 or over-expression of miR-376c-3p inhibited the viability of HeLa cells, induced apoptosis, enhanced the radiosensitivity, inhibited the protein expression of cyclin D and Bcl-2, and promoted the protein expression of P21 and Bax (P < 0.05). Linc00152 negatively regulated miR-376c-3p expression in the HeLa cells, and inhibition of miR-376c-3p expression reversed the effect of low expression of Linc00152 on HeLa cell viability, apoptosis and radiosensitivity. CONCLUSION: Linc00152 is highly expressed in the cervical cancer cells. Linc00152 affects the viability, apoptosis and radiosensitivity of HeLa cells by targeting miR-376c-3p, which is a potential diagnosis and treatment target for cervical cancer.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis

AIM To explore the effect of platelet-rich plasma (PRP) on rabbit osteoarthritis and its possible mechanism. METHODS The rabbits with knee osteoarthritis were prepared and then divided into model group, sodium hyaluronate (SH) group and PRP group, and another sham operation group was set up, with 6 rabbits in each group. The gross morphological changes of rabbit cartilage were observed. HE staining was used to evaluate the pathomorphological changes of the cartilage. TUNEL staining was used to detect the apoptosis of chondrocytes. The expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β （IL-1β） signaling pathway-related molecules was observed by immunohistochemical staining, and the protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. Chondrocytes were isolated and processed according to grouping, and the NLRP3 and IL-1β levels of the cells were measured by ELISA. RESULTS Compared with sham operation group, Pelletier score, Mankin score, chondrocyte apoptotic rate, the positive protein expression rates of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in model group were increased significantly (P<0.05), while the protein expression of Bcl-2 was decreased significantly (P<0.05). Compared with model group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly (P<0.05), while the protein expression of Bcl-2 was increased significantly (P<0.05). In PRP group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax were lower than those in SH group, while the protein expression of Bcl-2 was higher than that in SH group (P<0.05). Compared with control group, the expression of NL?RP3 and IL-1β in MCC950 (NLRP3 ihibitor) group were significantly reduced (P<0.05), the expression of NLRP3 in eucalyptol (IL-1β inhibitor) group was not significantly changed (P>0.05), and the expression of IL-1β was significantly reduced (P<0.05). CONCLUSION Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits, which has better effect than SH. The mechanism may be related to the inhibition of NLRP3/IL-1β pathway and the reduction of chondrocyte apoptosis.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

芜菁Br14-3-3的克隆及其在非生物胁迫下的表达分析

克隆了‘津田芜菁’（Brassica rapa subsp. rapifera‘Tsuda’）通用调节因子14-3-3基因cDNA序列,命名为Br14-3-3（GenBank登录号为MK896872）。该基因全长1 113 bp,开放阅读框全长774 bp,编码含有257个氨基酸的多肽。荧光定量PCR分析Br14-3-3在‘津田芜菁’不同组织中及其在温度、脱水、渗透、ABA和无机盐等非生物胁迫下幼苗中的表达,结果表明该基因在‘津田芜菁’的花中表达量最高,幼苗中次之;低温抑制了Br14-3-3的表达,其他非生物胁迫可诱导该基因表达,暗示Br14-3-3在非生物胁迫应答中发挥功能。     

云南、广西三七黑斑病病原链格孢菌的鉴定

正黑斑病是三七栽培生产中常见的一大病害,叶片受害产生近圆形或不规则水浸状病斑,常导致成株落叶、幼苗生长点及茎秆顶端腐烂枯死。其病原一般认为是链格孢属真菌人参链格Alternaria panax Whetzel~([1,2]),也有相关研究证明黑斑病病原为细链格孢Alternaria tenuis Nees~([3]),后定名为链格孢Alternaria alternata Keissl~([4])。本研究利用ITS序列和histone 3部分编码序列的PCR鉴定,结合形态学鉴定,分析三七主产区黑斑病菌的组成和分布情况及几种病原菌的致病力差异,以期为三七黑斑病防治提供理论依据。     

玉米轴色突变体698-3R的遗传鉴定

以辐射诱变获得的玉米红轴突变体698-3R为材料,通过遗传交配设计分析轴色的遗传规律,利用SSR-BSA法初步定位轴色基因,并对候选基因进行克隆和表达分析。结果表明:(1)突变体698-3R的红轴对白轴性状表现为显性遗传,受一对核基因控制,初步定位在第一染色体上的SSR标记phi095与umc1452之间,与phi095相距3.9cM,与umc1452相距7.1cM,将该基因暂定名为C(t);(2)以轴色基因 P1的cDNA为模板克隆C(t),发现698-3R有3个C(t)转录本,而野生型698-3中至少有2个;预测氨基酸序列比对发现,698-3R中3个C(t)蛋白的氨基酸序列与已知P_1-wr蛋白一致,突变使其获得完整的MYB结构域和酸性激活域,而野生型698-3中698-3-P-1蛋白由于编码序列缺失导致移码突变,不具有该功能结构域;(3)对C(t)基因qRT-PCR分析发现,除25DAP外,其余4个时期在698-3R中表达水平均显著或极显著高于698-3;以 A1和 C2基因表达验证分析发现,C(t)与验证基因表达模式一致,说明C(t)可能具有 P1激活调控 A1和 C2基因表达的功能。推测该红轴基因C(t)可能为一个 P_1-wr基因。