PI3K/Akt/GSK-3β pathway mediates ABC transporter regulating multidrug resistance in human colon cancer HCT-15 cells

AIM: To investigate whether the PI3K/Akt signaling pathway regulates the expression of ABC transporter through the downstream glycogen synthase kinase-3β (GSK-3β) pathway and participates in the multidurg resistance of colorectal cancer (CRC) HCT-15 cells. METHODS: Colorectal cancer HCT-15 cells were cultured and then treated with GSK-3β inhibitor (HY-19807) and PI3K/Akt pathway inhibitor (HY-13898), respectively. The median inhibitory concentration (IC₅₀) of oxaliplatin for HCT-15 cells in each group was detected by CCK-8 assay, the inhibition rate and resistance index were also calculated. The protein levels of Akt, p-Akt, GSK-3β, p-GSK3β-Ser9 and ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP-2) in the HCT-15 cells were determined by Western blot. The mRNA expression of ABC transporter in the HCT-15 cells was detected by RT-qPCR. The cell cycle distributions were analyzed by flow cytometry assasy. RESULTS: After GSK-3β inhibitor HY-19807 was used in the HCT-15 cells, the median inhibitory concentration of oxaliplatin was significantly increased, the protein levels of p-GSK3β-Ser9, P-gp and MRP-2 were up-regulated compared with control group (P<0.05), the changes of Akt and p-Akt were not obvious compared with control group (P>0.05). The results of RT-qPCR also showed that the mRNA levels of ABCB1 and ABCC2 were increased (P<0.01). Meanwhile, analysis of the cell cycle distribution showed that GSK-3β inhibitor HY-19807 promoted HCT-15 cell transition from G₁ phase to S phase, and cell proliferation was vigorous. After the PI3K/Akt pathway inhibitor HY-13898 was applied to HCT-15 cells, the IC₅₀ of oxaliplatin was decreased compared with control group (P<0.05). Moreover, the protein levels of p-Akt, p-GSK3β-Ser9, P-gp and MRP-2 were down-regulated (P<0.01). RT-qPCR results also showed that the mRNA expression of ABCB1 and ABCC2 was decreased (P<0.01). At the same time, G₁ phase was prolonged, which inhibited cell transition from G₁ phase to S phase, and inhibited cell proliferation. The protein expression of total GSK-3β was consistent in each group. CONCLUSION: The PI3K/Akt signaling pathway is involved in the proliferation and multidrug resistance of colorectal cancer HCT-15 cells by regulating the phosphorylation of GSK-3β and changing the expression of ABC transporter.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

PI3K/Akt signaling pathway regulates autophagy induced by acute kidney injury in septic rats

AIM：To investigate the autophagy induced by sepsis and acute kidney injury, and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process. METHODS：The rats were subjected to cecal ligation and puncture (CLP) or sham operation. Histopathologic changes of the renal tissues were examined by HE staining. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by chemical colorimetry. The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting. In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy. The protein expression of LC3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting. These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected. RESULTS：Compared with sham group, the severe changes of renal histopathological injuries in CLP groups were observed, the levels of BUN and SCr in CLP groups were significantly increased. LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP. After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose- and time-dependent fashion. The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment. Treatment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells. CONCLUSION：Autophagy in the kidney is induced by sepsis and acute kidney injury. PI3/Akt signaling pathway may be involved in this process.     

Xuebijing relieves testicular ischemia/reperfusion injury in rats by activating PI3K/Akt/mTOR signaling pathway

AIM: To investigate the effect of Xuebijing on testicular ischemia/reperfusion (I/R) injury in rats and its related mechanisms. METHODS: Male Sprague-Dawley rats (n=45) were randomly divided into control group, I/R group, low-dose Xuebijing group, high-dose Xuebijing group and dexamethasone group (n=9 in each group). Except for the rats in control group, the rats in other groups underwent testicular torsion, and after the operation, the rats were treated with 0.5 mL·kg^-1·d^-1 Xuebijing, 2 mL·kg^-1·d^-1 Xuebijing and 0.5 mL·kg^-1·d^-1 dexamethasone in low-dose Xuebijing group, high-dose Xuebijing group and dexamethasone group, respectively. On the 3rd, 7th, and 14th days after treatment, the left testis in the rats of each group was taken. The histopathological changes of the testis were observed by hematoxylin-eosin staining. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), endothelin-1 (ET-1) and nitric oxide (NO) in the testicular tissue were detected by biochemical methods. The protein levels of cell cycle-related molecules, apoptosis-related proteins and PI3K/Akt/mTOR signaling pathway-related proteins were determined by Western blot. RESULTS: Xuebijing significantly attenuated the testicular damage in I/R rats, significantly increased the activity of SOD in the testis of I/R rats, reduced the content of MDA, ET-1 and NO, inhibited oxidative stress in I/R-injured tissues, mediated the protein expression of cell cycle-related factors and apoptosis-related factors, and significantly increased the protein levels of p-PI3K, p-AKT, p-mTOR and p-S6K in the testis of I/R rats (P<0.05). These effects were time-dependent and dose-dependent. CONCLUSION: Xuebijing reduces testicular I/R injury of rats by mediating the expression of cell cycle-related and apoptosis-related proteins and activating PI3K/Akt/mTOR signaling pathway in dose-dependent and time-dependent manners.     

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.     

Effects of astragaloside IV combined with ginsenoside Rg1 on autophagy and PI3K signaling pathways of PC12 cells induced by oxygen glucose deprivation/reoxygenation

ATM: To probe the effect and the mechanism of astragaloside IV and ginsenoside Rg1 on autophagy of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The autophagy injury model of PC12 cells induced by OGD/R was established(PC12 cells were exposed to 2 h of OGD followed by 24 h of reoxygenation). The effects of astragaloside IV combined with ginsenoside Rg1 on autophagy of PC12 cells were observed, and the mechanism was studied through PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways. RESULTS: After OGD/R, LC3-Ⅱ/LC3-Ⅰin PC12 cells was increased. Astragaloside IV, ginsenoside Rg1 and astragaloside IV combined with ginsenoside Rg1 restrained the increase in LC3-Ⅱ/LC3-Ⅰ, the effect of the combination was greater than using the drug alone. Ginsenoside Rg1, astragaloside IV combined with ginsenoside Rg1 up-regulated the phosphorylation level of PI3K Ⅰ, Akt and mTOR. The effects of the combination were stronger than those of using the drug alone. Astragaloside IV, astragaloside IV combined with ginsenoside Rg1 inhibited the protein expression of PI3K Ⅲ and becline-1, the effects of the combination were better than those of single astragaloside IV and single ginsenoside Rg1. Meanwhile, the combination treatment increased Bcl-2 protein expression. CONCLUSION: The autophagy of PC12 cells induced by OGD/R is inhibited by astragaloside IV and ginsenoside Rg1. Furthermore, astragaloside IV combined with ginsenoside Rg1 plays synergitic inhibition on autophagy, the mechanism may be related to PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Adipophilin promotes intracellular lipid accumulation through PI3K/Akt signaling pathway

AIM:To observe the possible mechanism through which adipophilin promotes the accumulation of intracellular lipids, and to provide a reference for controlling atherosclerosis.METHODS:RAW264.7 cells were incubated with oxidized low-density lipoprotein (oxLDL) for different time. qPCR, Western blot and Oil red O staining were used to observe the mRNA and protein levels of Akt, p-Akt and adipophilin and lipid accumulation. The above indexes were measured after the cells were treated with PI3K/Akt signaling pathway inhibitor LY294002. The activation of Akt was analyzed in the HEK293 cells over-expressing adipophilin. Co-immunoprecipitation was applied for analysis of protein-protein interaction between adipophilin and Akt. RESULTS:After incubation with oxLDL, the amount of lipid droplets, Akt activity and adipophilin expression increased in the cells with the extension of time (P<0.05). Moreover, LY294002 inhibited the above changes. The p-Akt levels increased after adipophilin over-expression. No direct interaction between adipophilin and Akt proteins was observed. CONCLUSION:Adipophilin promotes the accumulation of intracellular lipids through PI3K/Akt signaling pathway, but possibly not by direct interaction between adipophilin and Akt proteins.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

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.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells

AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H₂O₂) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H₂O₂ group, 6-gingerol group (6-gingerol + H₂O₂) and LY294002 group (6-gingerol + H₂O₂ + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H₂O₂ at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H₂O₂ group were significantly increased compared with control group. The mitochondrial edema was obvious in H₂O₂ group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H₂O₂-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H₂O₂ induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H₂O₂-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Radiosensitizing effect of baicalin on human cervical carcinoma HeLa cells

AIM: To investigate the effect of baicalin on the radiosensitization of HeLa cells. METHODS: The cell activity was determined by MTT assay. The radiosensitivity of HeLa cells was detected by colony formation assay. The cell cycle was analyzed by flow cytometry. The protein levels of Akt, p-Akt, Bad and p-Bad were examined by Western blot. RESULTS: The cell growth of the HeLa cells was inhibited by baicalin dose-dependently and IC₅₀ was 43.65 mg/L. The results of colony formation assay showed that combination of 8 mg/L baicalin and radiotherapy further improved survival curve and decreased the value of D0 and Dq, as compared with radiotherapy alone (P<0.05). Furthermore, baicalin enhanced the effect of radiotherapy on cell cycle, as evidenced by the increase in cell percentage in G₂/M phase (P<0.05). Additionally, after incubation with baicalin, radiotherapy-induced phosphorylation of Akt and Bad were further augmented (P<0.05). CONCLUSION: Baicalin augments radiosensitivity of HeLa cells through the inhibition of cell cycle transition and activation of PI3K/Akt signaling pathway.