LBP regulates PI3K/Akt/eNOS signaling pathways in ovariectomized rat myocardium to exert antioxidative effect

AIM: To observe the influence of Lycium barbarum polysaccharide (LBP) on the PI3K/Akt/eNOS signaling pathways in ovariectomized rat myocardium. METHODS: Female SD rats (n=30) were divided into sham operation group, ovariectomized group, progynova group, high-dose LBP group and low-dose LBP group. The serum levels of estradiol, lactate dehydrogenase (LDH) and creatine kinase (CK) were measured by ELISA. The myocardial contents of H₂S and oxidative stress injury-related indicators were also detected. The morphological changes of the myocardium were observed with HE staining. The expression of eNOS and PI3K/Akt pathway-related proteins in the myocardium was determined by Western blot. RESULTS: Compared with sham operation group, the serum level of estradiol, the content of H₂S, the activity of GSH-Px, and the expression of eNOS and PI3K/Akt pathway-related proteins in the myocardium in ovariectomized group were all decreased, and the levels of ROS and MDA in the myocardium were increased (P<0.05). The serum levels of LDH and CK were also increased. The arrangement of the myocardial cells was disordered, and the intercellular space was also increased in the ovariectomized group. Compared with ovariectomized group, the serum level of estradiol, the myocardial levels of H₂S and GSH-Px, and the protein levels of eNOS and phosphorylated Akt were all increased in high dose group, while the levels of ROS and MDA in the myocardium were decreased (P<0.05). The serum levels of LDH and CK were also decreased. The morphological changes of the rat myocardium were improved in high dose group. CONCLUSION: LBP prevents and treats postmenopausal cardiovascular lesions through regulating PI3K/Akt/eNOS signaling pathways in ovariectomized rats.     

Hydrogen sulfide attenuates urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway

AIM:To explore the effect of hydrogen sulfide (H₂S) on urosepsis-induced acute kidney injury. METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, NaHS treatment (NaHS) group, and NaHS combined with TAK-242 (a TLR4 inhibitor) treatment (NaHS+TAK-242) group. After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer. The serum levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), procalcitonin (PCT), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot. RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with NaHS. The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-α in the septic rabbits were higher than those in control group, and decreased significantly in NaHS group and NaHS+TAK-242 group. The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group. However, NaHS or NaHS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits. CONCLUSION:H₂S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.     

Effects of hydrogen sulfide on impaired wound healing in ob/ob mice

AIM: To investigate the role of hydrogen sulfide(H₂S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS: The ob/ob mice were randomly divided into 3 groups, including vehicle, insulin and NaHS for treatment. C57BL/6 mice were treated with vehicle as control. Full-thickness punch biopsy wounds were created on the mice. Firstly, H₂S concentrations in the skins and granulation tissues were measured. The mRNA expression of cystathionine γ-lyase(CSE) was detected by RT-qPCR. The protein expression of CSE and MMP-9 were determined by Western blot. The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod. The levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS: The H₂S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice(P<0.05). CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice(P<0.05). The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group(P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group. The neutrophil and monocyte/macrophage infiltration, and TNF-α and IL-6 levels were significantly increased in ob/ob groups, but were decreased in NaHS group(P<0.01 or P<0.05). Meanwhile, NaHS increased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION: H₂S/CSE down-regulation contributes to impaired wound healing in diabetes, which is alleviated by exogenous H₂S possibly through anti-inflammation.     

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.     

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.     

Role of hydrogen sulfide in acute lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM: To explore the role of endogenous and exogenous hydrogen sulfide (H₂S) in acute lung injury (ALI) induced by ischmia-reperfusion (IR) of hind limbs in rats.METHODS: A Sprague-Dawley rat model of acute lung injury was induced by ischemia of the hind limbs for 4 h and reperfusion for another 4 h. The rats (n=120) were randomly divided into 4 groups: control, IR, NaHS (H₂S donor)+IR, and propargylglycine +IR. The animals were sacrificed after reperfusion. Lung weight/body weight ratio (LW/BW) was measured and calculated. Morphological changes of the lung tissues were observed. The concentrations of H₂S, nitric oxide (NO) and carbon monoxide (CO) in plasma were tested. The content of malondialdehyde (MDA), the activity of CSE, inducible nitric oxide synthase (iNOS) and hemeoxygenase (HO) in the lungs were determined. The polymorpho-nuclear neutrophils(PMN) and protein content in bronchoalveolar lavage fluid(BALF) were also measured. The correlation of H₂S content with the above indices was analyzed.RESULTS: Compared with control group, severe injuries of the lung tissues, raised LW/BW, MDA concentration, PMN and protein contents in BALF were observed in IR group. Limb IR also made a drop in the concentration of plasma H₂S and the activity of lung CSE, while the activity of iNOS and HO in the lung tissues and the levels of plasma NO and CO increased. Administration of NaHS before IR attenuated the changes induced by IR, while pre-administration of PPG exacerbated the IR injuries and increased the plasma NO level and lung iNOS activity. The H₂S content was positively correlated with CSE activity, CO content and HO-1 activity (P<0.01), and negatively correlated with the other indices (P<0.01).CONCLUSION: Down-regulation of H₂S/CSE is involved in the pathogenesis of acute lung injury induced by IR. Endogenous and exogenous H₂S protects against lung injuries. The anti-injury effects of H₂S are related with its anti-oxidative activity to attenuate the inflammatory over-reactions in the lung induced by PMN. Down-regulation of NO/iNOS system and up-regulation of CO/HO-1 system by H₂S are also involved in the process of anti-injury to ALI.     

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.     

Exogenous hydrogen sulfide attenuates obstructive kidney injury in mice

AIM:To investigate the protective effect of exogenous hydrogen sulfide (H₂S) on obstructive renal injury in mice, and to explore the possible potential mechanisms involved in this animal model. METHODS:Male C57BL/6 mice (8 weeks old) were randomly divided into sham group, operation group and H₂S group, with 5 rats in each group. The model of obstructive renal injury was induced by unilateral ureteral obstruction (UUO). The mice in H₂S group were intraperitoneally injected with NaHS daily, while the mice in sham group and operation group were administered with the same volume of saline intraperitoneally. After 7 d, the mice were executed and the renal tissues were taken out for experiments. RNA was extracted to detect the mRNA expression of H₂S catalytic enzymes in the mice of 3 groups. HE staining was performed to observe the structural changes of renal tissues in the mice. Renal fibrosis in the mice of 3 groups was evaluated by Masson staining. The content of cystatin C in the plasma was detected to reflect glomerular filtration ability. The protein expression of LC3, beclin-1 and fibronectin (FN) in the mice of 3 groups was determined by Western blot. RESULTS:Compared with sham group, the mRNA expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) in operation group decreased significantly. The collagen fiber content in operation group was increased significantly, while collagen fiber content in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of FN in operation group was increased significantly, while the protein expression of FN in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of LC-Ⅱ and beclin-1 in operation group was increased significantly, while the protein expression of LC-Ⅱ and beclin-1 in H₂S group was increased significantly as compared with the operation group. CONCLUSION:Exogenous H₂S possibly mitigates renal fibrosis in UUO mice by up-regulating autophagy.     

Adiponectin activates AKT and protects rat cardiomyocytes against H2O2-induced apoptosis

AIM: To study the effect of adiponectin on H₂O₂-induced apoptosis in rat cardiomyocytes (H9c2 cells). METHODS: Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was used to determine H₂O₂-induced apoptosis of H9c2 cells in the absence or presence of adiponectin. The content of caspase-3 and Akt (protein kinase B, PKB) was examined by Western blotting. RESULTS: Adiponectin significantly inhibited H₂O₂-induced apoptosis in H9c2 cells (P<0.01). Adiponectin increased basal and H₂O₂-induced Akt activity and significantly inhibited H₂O₂-induced activation of caspase-3 (P<0.01). Pretreatment with LY294002, a specific inhibitor of Akt upstream kinase PI3K (phosphatidylinositol 3-kinase), not only increased H₂O₂-induced H9c2 cell apoptosis, but also abrogated the anti-apoptotic effect of adiponectin. CONCLUSION: Adiponectin protects H9c2 cells against H₂O₂-induced apoptosis by activating PI3K/Akt signaling pathway.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

Endogenous hydrogen sulfide regulates oxidant/antioxidant balance in rat with chronic obstructive pulmonary disease

AIM: To investigate the role of hydrogen sulfide (H₂S) in the protection against oxidative stress in rats with chronic obstructive pulmonary disease (COPD).METHODS: The rat model of COPD was established by cigarette smoking (CS) combined with lipopolysaccharide (LPS) instillation. Thirty-two healthy male Sprague-Dawley rats were randomly divided into 4 groups: control group, CS+LPS group, CS+LPS+NaHS (H₂S donor) group and CS+LPS+PPG (DL-propargylglycine, an inhibitor of cystathionine-γ-lyase) group. After 30 days, the lung functions of the rats were measured, the histological changes of lungs were observed under light microscope and the pathological scores were calculated. The H₂S level in plasma and the protein expression of cystathionine-γ-lyase (CSE) in the lung tissues were measured. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and catalase (CAT) were detected to reflect oxidative stress.RESULTS: Compared with control group, the peak expiratory flow (PEF) decreased by 24% and intra-pressure (IP) increased by 66% in CS+LPS group. The pathological scores of the lung tissues also increased. Compared with CS+LPS group, no change in the lung function was observed after given NaHS or PPG, but the pathological scores decreased in CS+ LPS+ NaHS group. Compared with control group, the content of H₂S in plasma was increased by 26% on day 16. Compared with CS+LPS group, the content of H₂S in plasma of CS+LPS+PPG group was decreased by 22% after 30 days. Compared with control group, the protein expression of CSE increased, and no statistical difference among CS+LPS group, CS+ LPS+ NaHS group and CS+LPS+PPG group was observed. Compared with control group, MDA content in the lung tissues was increased by 24% in CS+LPS group, the activity of SOD was increased by 47% and the activity of CAT was increased by 52%. Compared with CS+LPS group, the MDA content in CS+LPS+NaHS group was decreased by 21%, and no statistical difference in the activity of SOD and CAT was observed. The activity of SOD decreased by 33% after given PPG.CONCLUSION: H₂S plays a role as anti-oxidant in the rats with COPD. The CSE/ H₂S pathway may be involved in the development of COPD.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

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

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

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.     

NVP-BEZ235 induces autophagy of polycystic kidney rat cholangiocytes

AIM: To explore the effect of dual PI3K/Akt/mTOR inhibitor NVP-BEZ235 on autophagy of polycystic kidney (PCK) rat cholangiocytes. METHODS: The protein levels of p-mTOR and p-Akt in the bile duct epithelial cells were examined by immunohistochemistry. The effect of NVP-BEZ235 on the viability of cholangiocytes was detected by WST-1 assay. The levels of PI3K/Akt/mTOR signaling pathway and autophagy-related proteins with NVP-BEZ235 treatment were determined by Western blot. The effects of LC3 and Beclin 1 silencing, and authophagy-specific inhibitor 3-methyladenine (3-MA) on the cell viability were analyzed by WST-1 assay. RESULTS: The protein levels of p-Akt and p-mTOR were highly increased in the bile duct epithelium of the PCK rats. NVP-BEZ235 significantly inhibited the viability of the cholangiocytes in a dose- and time-dependent manner (P<0.05). NVP-BEZ235 significantly reduced the levels of PI3K/Akt/mTOR signaling pathway-related proteins in the PCK rat cholangiocytes. NVP-BEZ235 upregulated the autophagy-specific proteins LC3 II and Beclin 1. The inhibitory effect of NVP-BEZ235 on the cell viability was weakened by treatment with 3-MA and knockdown of LC3 and Beclin 1 (P<0.01).CONCLUSION: The PI3K/Akt/mTOR inhibitor NVP-BEZ235 suppresses the viability of PCK rat cholangiocytes, and the mechanism is closely related with autophagy.     

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.     

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.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.     

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.