Inhibitory effect of ATP on proliferative signaling in immortalized human fibroblasts

AIM: To investigate the inhibitory effects of ATP on proliferation signaling in immortalized human fibroblasts. METHODS: Immortalized human fibroblasts were treated with ATP, ATP conbined with calcium or potassium channel antagonists, respectively. The intracelluar-free calcium ([Ca²⁺]i), inositol 1,4,5-trisphosphate(IP₃) levels and cell viability were detected at different time points. RESULTS: ATP significantly increased the [Ca²⁺]i and decreased the IP₃ level in immortalized human fibroblasts, especially at initial stage (P＜0.01) . Compared to ATP alone, the proliferation rates remarkably increased when calcium or potassium channel antagonists were used (P＜0.01, respectively) with ATP. CONCLUSION: The calcium and potassium channels and IP₃ involved in the inhibitory effects of ATP on the proliferative signaling in immortalized human fibroblasts.     

Qishen-Yiqi dripping pills attenuate atherosclerosis by regulating Ca2+ homeostasis via TRPC1/STIM1 pathway

AIM:To explore the therapeutic effect of Qishen-Yiqi dripping pills (QS) on atherosclerosis (AS) and the mechanism. METHODS:AS rat model was established by high-fat diet, and SD rats were randomly divided into normal control group, AS model group, low-dose, middle-dose and high-dose QS groups, and positive group (n=6 each). After administration for 12 weeks, serum samples were collected to detect the serum lipid and Ca²⁺ levels. HE staining was used evaluated the histopathological changes of arterial tissue. The serum levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. The nitric oxide (NO) level was detected by nitrate reductase method. The protein levels of transient receptor potential channel protein 1 (TRPC1), stromal interaction molecule 1 (STIM1) and endothelial NO synthase (eNOS) were determined by Western blot. RESULTS:QS significantly reduced the arterial damage via inhibiting the formation of atherosclerotic plaque and attenuated intimal thickening and vascular stenosis. Compared with AS group, the serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were decreased significantly and the levels of high-density lipoprotein cholesterol (HDL-C) were increased significantly in high-dose QS group (P<0.05). The serum levels of IL-1β, IL-6 and TNF-α in high-dose QS group were lower than those in AS group (P<0.05). Compared with AS group, the serum Ca²⁺ level was lowered and the arterial tissue NO level was elevated in QS groups (P<0.05). Compared with AS rats, the protein levels of TRPC1 and STIM1 were decreased significantly and the protein level of eNOS was increased significantly in the rats treated with QS (P<0.05). CONCLUSION:QS regulate calcium homeostasis via TRPC1/STIM1 pathway, increase the production of NO and inhibit the inflammatory responses, thus exerting anti-AS effect.     

Role of phosphoinositide pathway in the formation of cardiac hypertrophy induced by pressure overload in rats

AIM: To investigate the role of phosphoinositide pathway in the formation of pressure-overload cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced in male Sprague-Dawley rats with coarctation of abdominal aorta, whole heart weight/body weight ratio was tested after 10 or 30 days of operation. Content of Gαq/11 protein in left ventricle was detected by immunoblot analysis and concentration of IP₃ was measured by radioimmunoassay. RESULTS: At 10 and 30 days, whole heart weight/body weight ratio of coarctation aorta (CA) group was higher than that of sham-operated (SO) rats (P＜0.01). Protein Gαq/11 contents were not modified after 10 or 30 days of stenosis (P＞0.05). At 10 days, the level of IP₃ significantly increased in left ventricle of CA rats compared with the control animals (P＜0.05), but there was no difference in IP₃ level between CA and SO group after 30 days of operation. CONCLUSION: Phosphoinositide signaling pathway may play a role in the early stage of cardiac hypererophy induced by pressure overload.     

Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

Doxorubicin induces stemness of mouse TNBC 4T1 cells through Stat3-Oct-4/CD44 signaling pathway

AIM:To investigate the stemness of mouse triple-negative breast cancer (TNBC) 4T1 cells induced by doxorubicin (DOX) and the underlying mechanism. METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations (0, 0.05, 0.1 and 0.5 μmol/L) for 24 h, and the shape and viability of the cells were observed. The concentration of DOX at 0.1 μmol/L was chosen as the optimal concentration for the following experiments. The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks, and named as 4T1-DOX and MDA-MB-468-DOX. Sphere formation assay was used to detect the stemness of 4T1 cells and MDA-MB-468 cells. The expression of CD133 was observed by immunofluorescence staining. The expression of CD44 was analyzed by flow cytometry. The protein levels of Stat3, phosphorylated Stat3 (p-Stat3) and Oct-4 were determined by Western blot. RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1 control cells. The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4T1 cells (P<0.05). Furthermore, the 4T1-DOX cells exhibited enhanced activation of Stat3 (p-Stat3) and increased expression of Oct-4 (P<0.05), while the expression of total Stat3 had no obvious variation. In addition, when activation of Stat3 was inhibited by WP1066, the protein levels of p-Stat3, Oct-4 and CD44 were down-regulated (P<0.05). Furthermore, inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells (P<0.05). CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

Role of miR-433 in fibrosis of NIH-3T3 cells and silica-induced pulmonary fibrosis in mice

AIM To study the role and regulatory mechanism of microRNA-433 (miR-433) in fibrosis. METHODS TargetScan was used to predict the potential target genes of miR-433. The changes of miR-433 expression were detected after transforming growth factor β1 (TGF-β1) treatment of mouse embryonic fibroblasts (NIH-3T3 cells) for 24 h. The effects of miR-433 mimic on the expression of p-SMAD2, fibronectin (FN), α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in TGF-β1 treated cells were examined. The effects of miR-433 mimic transfection on the viability and S-phase fraction of NIH-3T3 cells induced by TGF-β1 were detected by CCK-8 assay and flow cytometry. A model of silica-induced pulmonary fibrosis in mice was established, and agomiR-433 was used for intervention. HE staining and Masson staining were used to observe the effect of miR-433 on pulmonary fibrosis in the mice. The expression of α-SMA in lung tissues was detected by immunohistochemistry. RESULTS miR-433 specifically bound to the 3'-UTR of SMAD2 and inhibited its expression at protein and mRNA levels. TGF-β1 down-regulated the expression of miR-433 in NIH-3T3 cells, up-regulated the protein level of p-SMAD2 and the expression of FN, α-SMA and CTGF at protein and mRNA levels, and increased the viability and the number of S-phase cells. miR-433 mimic reversed the effects of TGF-β1 on NIH-3T3 cell viability and S-phase arrest. In a model of silica-induced pulmonary fibrosis in mice, agomiR-433 inhibited the progress of pulmonary fibrosis and reduced the expression of α-SMA in mouse lung tissues. CONCLUSION miR-433 may interfere with TGF-β1/SMAD2 signaling pathway through targeting SMAD2, thus participating in the regulation of fibrosis process.     

Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous H2S production aggravate intestinal injury in severe acute pancreatitis

AIM:To investigate the potential role of endogenous hydrogen sulphide (H₂S) in severe acute pancreatitis (SAP). METHODS:A rat model of SAP was used to evaluate the role of H₂S on intestinal motility by counting the number of fecal pellets and the effect of H₂S on the expression of inflammation-related molecule in intestine was investigated. The colonic muscle cells (CMCs) were treated with plasma of SAP rats, tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6), and the expression of cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), Sp1 and PI3K/Akt related proteins at mRNA and protein levels were determined by RT-qPCR, Western blot and immunohistochemical staining,respectively. The PI3K inhibitor LY294002 and the siRNA-Sp1 were used to suppress the activity of PI3K/Akt/Sp1 signaling pathway. RESULTS:H₂S facilitated an inhibitory effect on the intestinal motility and enhanced the inflammatory responses in SAP (P<0.05). The expression of CSE and CBS in CMCs was significantly increased after treatment with TNF-α or IL-6 (P<0.05). Blockage of the PI3K/Akt/Sp1 signaling pathway remarkably inhibited the synthesis of CSE and CBS in CMCs(P<0.05). CONCLUSION:Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous production of H₂S play a vital role in the pathogenesis of SAP.     

Effects of PI3K/PKB signaling pathway on expression of osteopontin in human hepatic stellate cells induced by transforming growth factor-β1

AIM: To investigate the regulatory effects of phosphatylinositol 3-kinase/protein kinase B (PI3K/PKB) signaling pathway on the expression of osteopontin (OPN) in transforming growth factor-β₁ (TGF-β₁)-induced human hepatic stellate cells. METHODS: Human hepatic stellate cell line LX-2 was cultured in DMEM and stimulated by TGF-β₁ at the final concentration of 2.5, 5, 10 and 20 μg/L for 24 h or at final concentration of 10 μg/L for 12 h, 24 h and 48 h. LX-2 cells were pretreated with wortmannin, a specific inhibitor of PI3K/PKB signaling pathway, at final concentration of 0.1 μmol/L for 1 h, followed by incubation with TGF-β₁ at final concentration of 10 μg/L for 24 h. The cells were collected. The expression of OPN was detected by real-time PCR and Western blotting. RESULTS: In LX-2 cells, the expression of OPN was apparently elevated when incubated with TGF-β₁. With the increase in TGF-β₁ concentration or the extension of incubation hours, the expression of OPN was increased gradually in a dose-and time-dependent manner with certain limits. LX-2 cells pretreated with wortmannin and incubated with TGF-β₁ had a significant decrease in the OPN expression as compared with control group (P<0.01). CONCLUSION: The expression of OPN in TGF-β₁-induced LX-2 cells is regulated by the PI3K/PKB signaling pathway.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Acute cerebral ischemia activates EphB2/ephrin-B1/NMDA receptor signaling pathway to promote hippocampal neurogenesis in mice

AIM To investigate the effect of acute cerebral ischemia on hippocampal neurogenesis in mice and its possible mechanism involving EphB2/ephrin-B1/NMDA receptor signaling pathway. METHODS C57BL/6 mice (n=52) were randomly divided into sham group and acute cerebral ischemia group (model group). The model of acute cerebral ischemia in mice was established by bilateral common carotid artery occlusion. The pathological changes of the hippocampal CA1 region in mice were observed by HE staining. The learning and memory functions of the mice were assessed by Morris water maze. The BrdU positive cells and doublecortin (DCX) protein expression were observed by immunofluorescence staining for detecting hippocampal neurogenesis. The mRNA and protein expression levels of EphB2, ephrin-B1, reelin, microtubule-associated protein-2 (MAP-2) and NMDA receptor subunits NR2A and NR2B in the hippocampus were determined by RT-qPCR and Western blot. RESULTS The neuronal damage in the hippocampal CA1 region was significant (P<0.01), and the learning and memory functions were significantly decreased in the cerebral ischemia mice(P<0.01), suggesting that the cerebral ischemia model was successfully established. The BrdU positive cells and DCX protein expression were increased significantly (P<0.01), indicating that neurogenesis occurred in the hippocampus after cerebral ischemia. At the same time, the mRNA and protein expression levels of EphB2, ephrin-B1, reelin, MAP-2, NR2A and NR2B in the hippocampus were also significantly up-regulated (P<0.05). CONCLUSION Acute cerebral ischemia promotes the proliferation of hippocampal neural stem cells and endogenous neurogenesis, which may be related to the activation of EphB2/ephrin-B1/NMDA receptor signaling pathway.     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

AIM: To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process. METHODS: The replicative senescence model of primary human umbilical vein endothelial cells (HUVECs) was established. The morphological change of the cells, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed. The NO concentration in the cell culture supernatant was measured by nitrate reductase assay. RESULTS: HUVECs with cumulative population-doubling level (CPDL) at 16 were chosen as the replicative senescence model in this research. Ginsenoside Rb1 at 80 μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels. Furthermore, ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels, and increased the NO content. SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation, leading to an increase in the expression of PAI-1 at mRNA and protein levels. Upon intervention of ginsenoside Rb1, the eNOS and PAI-1 expression and the level of NO were not reversed. CONCLUSION: Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

let-7a inhibits MAP4K3/MKK4/JNK signaling pathway to decrease neuronal apoptosis in rats with intracerebral hemorrhage

AIM To investigate the effect of microRNA let-7a on neuronal apoptosis in a rat model of intracerebral hemorrhage (ICH). METHODS Forty-eight healthy SD rats were selected, 8 of which served as sham group, and 2 μL of type VII collagenase was injected into globus pallidus of the remaining rats to construct ICH model. These ICH rats were randomly divided into model (2 μL normal saline) group, let-7a agomir group, NC agomir group, let-7a antagomir group and NC antagomir group, with 8 rats in each group, and the corresponding drugs were injected into the lateral ventricle. After 7 d, the neurological function scoring was performed, and HE staining was used to observe the pathological damage. RT-qPCR and Western blot were used to detect the expression of related proteins. TUNEL staining was used to detect neuronal apoptosis. The targeting relationship between let-7a and mitogen-activated protein kinase kinase kinase kinase 3 （MAP4K3） was predicted by bioinfomatic software, and further verified by dual-luciferase reporter assay. RESULTS In animal experiments, when let-7a was over-expressed, neurological function scores were reduced,and the pathological damage was alleviated. Glial fibrillary acidic protein (GFAP) expression was down-regulated, neuronal apoptosis was reduced, and the protein levels of cleaved caspase-3 and cleaved PARP were decreased (P<0.05). In cell assays, MAP4K3 was one of the target genes of let-7a, and MAP4K3 was negatively regulated by let-7a. let-7a over-expression inhibited the expression of MAP4K3/MKK4/JNK. CONCLUSION let-7a reduces neuronal apoptosis in ICH rats by inhibiting the MAP4K3/MKK4/JNK signaling pathway.