Clearance effect of regulation of serine/threonine protein kinase-1 signaling pathway by insulin on pulmonary edema in mice with acute lung injury / 中国生物制品学杂志

@#Objective To investigate the mechanism of insulin alleviating pulmonary edema in mice with acute lung injury(ALI) by serine/threonine protein kinase-1(SGK1).Methods 32 male adult C3H/HeN mice were randomly divided into control group(only pumped with the same amount of normal saline as the treatment group),ALI group(continuously pumped with the same amount of normal saline as the treatment group after modeling),treatment group [continuously pumped with 0.1 U/(kg·h) of insulin through jugular vein after establishing ALI model] and SGK1 siRNA group[continuously pumped with 0.1 U/(kg·h) of insulin and given SGK1 siRNA(75 μg SGK1 siRNA diluted in 100 μL saline) simultaneously after establishing ALI model] with 8 mice in each group.After 8 h,the mice were killed for arterial blood gas analysis(1 h after establishment of the model) and the changes of plasma glucose levels were detected(0,1,4and 8 h);The bronchoalveolar lavage fluid(BALF) was collected to detect the content of total protein,and the alveolar epithelial permeability and lung water content were measured;The pathological changes of lung tissue and apoptosis of lung epithelial cells were observed;The protein expressions of alveolar epithelial sodium channel(ENaC) and α_1-Na~+,K~+-ATPase and the phosphorylation level of SGK1 were determined by Western blot.Results There was no significant difference in plasma glucose level of ALI and treatment group at 0,1,4 and 8 h after insulin infusion(t=1.330 0,0.986 0,0.565 7 and 0.724 3,P=0.204 7,0.340 7,0.580 6 and 0.480 8,respectively).Compared with ALI group,the partial pressure of oxygen in arterial blood in treatment group increased significantly(t=6.026,P <0.000 1),while the BALF protein content,alveolar epithelial permeability,lung water content and lung epithelial cells apoptosis decreased significantly(t=7.39,5.286,5.651 and 3.312,P <0.000 1,=0.000 4,=0.000 2 and=0.007 8,respectively),and the expression of α-ENaC and α_1-Na~+,K~+-ATPase and the phosphorylation level of SGK1 in lung tissue significantly increased(t=26,18.67 and 8.547,P <0.000 1,<0.000 1 and=0.000 1,respectively);Compared with the treatment group,the BALF protein content,alveolar epithelial permeability,lung water content and lung epithelial cells apoptosis increased significantly in SGK1 siRNA group(t=5.964,3.449,3.148 and 3.520,P=0.000 2,0.006 2,0.010 4 and0.016 9,respectively),while α-ENaC and α_1-Na~+,K~+-ATPase protein expression and SGK1 phosphorylation level decreased significantly(t=13,9.874 and 7.741,P <0.000 1,<0.000 1 and=0.001 5,respectively).Conclusion Exogenous insulin can alleviate the pulmonary edema in ALI mice,which might be mediated via up-regulation of the expressions of α-ENaC and α_1-Na~+,K~+-ATPase by SGK1.

Eplerenone protects contralateral kidney tissue from obstruction in pregnant rats with chronic kidney disease / 中国药理学通报

Aim To study the protective effect of eplerenone on the contralateral kidney in pregnant rats with chronic kidney disease (CKD) and its mechanism. Methods Female Wistar rats were randomly divided into sham-operation group, sham-operation pregnancy group, model group and eplerenone group. The rats in the model group and eplenone group had ligation unilateral ureter, and the rats in the eplenone group were treated with 100 mg • kg

Overexpressed SIRT6 ameliorates doxorubicin-induced cardiotoxicity and potentiates the therapeutic efficacy through metabolic remodeling

Since the utilization of anthracyclines in cancer therapy, severe cardiotoxicity has become a major obstacle. The major challenge in treating cancer patients with anthracyclines is minimizing cardiotoxicity without compromising antitumor efficacy. Herein, histone deacetylase SIRT6 expression was reduced in plasma of patients treated with anthracyclines-based chemotherapy regimens. Furthermore, overexpression of SIRT6 alleviated doxorubicin-induced cytotoxicity in cardiomyocytes, and potentiated cytotoxicity of doxorubicin in multiple cancer cell lines. Moreover, SIRT6 overexpression ameliorated doxorubicin-induced cardiotoxicity and potentiated antitumor efficacy of doxorubicin in mice, suggesting that SIRT6 overexpression could be an adjunctive therapeutic strategy during doxorubicin treatment. Mechanistically, doxorubicin-impaired mitochondria led to decreased mitochondrial respiration and ATP production. And SIRT6 enhanced mitochondrial biogenesis and mitophagy by deacetylating and inhibiting Sgk1. Thus, SIRT6 overexpression coordinated metabolic remodeling from glycolysis to mitochondrial respiration during doxorubicin treatment, which was more conducive to cardiomyocyte metabolism, thus protecting cardiomyocytes but not cancer cells against doxorubicin-induced energy deficiency. In addition, ellagic acid, a natural compound that activates SIRT6, alleviated doxorubicin-induced cardiotoxicity and enhanced doxorubicin-mediated tumor regression in tumor-bearing mice. These findings provide a preclinical rationale for preventing cardiotoxicity by activating SIRT6 in cancer patients undergoing chemotherapy, but also advancing the understanding of the crucial role of SIRT6 in mitochondrial homeostasis.

Ligustrazine suppresses platelet aggregation through inhibiting the activities of calcium sensors

Abstract Ligustrazine is widely used for the treatment of cardiovascular diseases in traditional Chinese medication. It has been reported that Ligustrazine decreases the concentration of intracellular calcium ions (Ca2+); however, the underlying mechanism remains unknown. In the present study, the effect of Ligustrazine on adenosine diphosphate (ADP)-induced platelet aggregation was evaluated using a turbidimetric approach. The changes in concentration of intracellular Ca2+ stimulated by ADP was measured using fluo-4, a fluorescent Ca2+ indicator dye. The mRNA expression of stromal interaction molecule l (STIM1) and Orai1, calcium sensor, was determined using real-time PCR. In addition, the protein expression of STIM1, Orai1, and serum/glucocorticoid-regulated protein kinase 1 (SGK1) was determined using Western blot analysis. The data demonstrated that Ligustrazine significantly suppressed platelet aggregation in a dose-dependent manner and reduced the concentration of intracellular Ca2+ triggered by ADP. Our data showed that Ligustrazine treatment inhibited the expression of STIM1 and Orai1 induced by ADP at both mRNA and protein levels, and suppressed the protein expression of SGK1. Taken together, our data indicated that Ligustrazine suppressed platelet aggregation by partly inhibiting the activities of calcium sensors, thereby suggesting that Ligustrazine may be a promising candidate for the treatment of platelet aggregation.

Inhibition of mineralocorticoid receptor activation by eplerenone to reduce autophagy in obstructive nephropathy / 中国药理学通报

Aim To observe the effect of mineralocorticoid receptor blocker eplerenone on autophagy in obstructive nephropathy and its mechanism. Methods Totally 36 male Wistar rats were randomly divided into sham-operation group, model group and eplerenone group. The animal models were established with unilarteral urteral obstruction ( UUO). The rats in eplerenone group were treated with eplerenone (100 mg • kg"1 • d"1). The obstructed kidneys were collected lOd after UUO. The expression of NR3C2 was detected by laser confocal microscopy, the expression of serum and glucocorticoid-induced protein kinase 1 ( SGK-1), phosphorylated mammal target of rapamycin (p-mTOR) , autophagy associated gene 5 (Atg5) , Be-clin-1 and microtubular-associated protein 1 light chain 3 (LC3) were detected by immunohistochemistry and Western blot. Results The expression of NR3C2 was detected in cytoplasm of renal tubular distal epithelial cells, but not in nucleus in sham-operation group with laser confocal microscopy. The expression of NR3C2 was enhanced significantly in model group, mainly in nucleus but significantly inhibited in eplerenone group. The immunohistochemistry and Western blot showed that the expressions of SGK-1, Atg5, Beclin-1 and the ratio of LC3 11/I in model group were up-regulated and down-regulated by eplerenone treated group. The expression of p-mTOR was down-regulated in model group compared with sham-operation group and up-regulated in eplerenone group. Conclusions Eplerenone plays a role in reducing autophagy in obstructive nephropathy via inhibiting the activation of mineralocorti-coid receptor.

Mechanism of Shenluotong regulating NR3C2/SGK-1/Smad pathway in inhibiting renal interstitial fibrosis / 中草药

Objective To investigate the mechanism of Shenluotong inhibiting renal interstitial fibrosis by regulating NR3C2/SGK-1/Smad pathway. Methods A total of 48 Wistar rats were randomly divided into sham operation group, model group, Eplerenone group, and Shenluotong group (n = 12). Model group, Eplerenone group, and Shenluotong group used unilateral ureteral obstruction (UUO) method to establish rat renal interstitial fibrosis model. After the operation, the rats in the eplerenone group were treated with eplerenone at a dose of 100 mg/(kg∙d). Rats in the Shenluotong group were oral given Shenluotong decoction at a dose of 26 g/(kg∙d) and Sham operation group and model group were administrated equal volume of saline once daily for continuous 10 d. Laser confocal microscopy was used to detect mineralocorticoid receptor NR3C2 expression. The expressions of SGK-1, TGF-β1, Smad4, and Smad7 in renal tissues were detected by immunohistochemistry, Western Blot and Real time-PCR. Results In the sham operation group, NR3C2 was expressed in the cytoplasm of renal tubular epithelial cells and was not expressed in the nucleus. The expression of NR3C2 in the UUO rat was significantly up-regulated in cytoplasm and positive expression was observed in the nucleus. The expression of NR3C2 in the nucleus of cells in the Eplerenone group and Shenluotong group was significantly decreased when compared with the model group. Compared with the sham-operated group, the expression of SGK-1, TGF-β1, and Smad4 was significantly up-regulated and the expression of Smad7 was significantly decreased (P < 0.05, 0.01) in the other groups. Compared with model group, the expression range and intensity of TGF-β1 and Smad4 were significantly decreased in Eplerenone group and Shenluotong group (P < 0.05, 0.01), and the expression range and intensity of Smad7 were significantly increased (P < 0.01). Conclusion Shenluotong can inhibit renal interstitial fibrosis through blocking the activation of mineralocorticoid receptor, reducing the level of SGK-1, and regulating the Smads signal pathway to inhibit the overexpression of TGF-β1.

Effects of Baishile Capsules on Hippocampal PI3K Signaling Pathway in Chronic Mild Unpredictable Stress Rats / 中国中医药信息杂志

Objective To observe the effects of Baishile Capsules on the hippocampal PI3K signaling pathway in chronic mild unpredictable stress (CUMS) rats; To discuss its mechanism of action for anti-depression. Methods SD rats were randomly divided into 6 groups, control group, model group, fluoxetine group, Baishile Capsules high-, medium- and low-dose groups, with 10 rats in each group. The cums depression model was established, and gavage for medication was conducted at the same time for 21 days in a row. The behavioral changes of rats in each group were detected by the novel feeding experiment and Open-field experiment; monoamine neurotransmitter in serum were detected by ELISA; the expressions of PI3K, AKT and SGK1 in rat hippocampus were detected by immunofluorescence and Western blot test. Results Compared with the control group, feeding latency of rats in model group was significantly prolonged (P<0.01), horizontal and vertical activities were significantly reduced (P<0.01); the content of serum monoamine neurotransmitter decreased (P<0.01); the expressions of PI3K and AKT in hippocampus decreased, while the expression of SGK1 increased (P<0.01, P<0.05). Compared with the model group, Baishile Capsule high- and medium-dose groups can significantly shorten the incubation period of feeding rats (P<0.05), and increase the number of horizontal and vertical activities (P<0.01); the 5-HT and NE levels in serum were significantly elevated (P<0.05, P<0.01); the expressions of PI3K, AKT in hippocampal increased; the expression of SGK1 decreased (P<0.05, P<0.01). Conclusion Baishile Capsules can alleviate the depression like behavior in rat models, and regulate key factors of hippocampal PI3K signaling pathway, so as to exert antidepressant effects.

Resveratrol up-regulates the expression of alveolar epithelialsodium channel in acute lung injury mice via activation of SGK1 / 基础医学与临床

Objective To investigate the effect of resveratrol on alveolar epithelial sodium channel in acute lung injury mice and the potential mechanism.Methods Twenty-four C57BL/6 mice were randomly divided into control group, LPS group, RES group and PP242(mTORC inhibitor) group with 6 mice in each group.The pathological changes in lung tissue were evaluated by HE staining;the concentrations of total protein in bronchoalveolar lavage fluid (BALF) were assessed by BCA (bicinchoninic acid).The levels of inflammatory cytokines in BALF were determined by ELISA.The proportions of polymorphonuclear neutrophil (PMN) in BALF were detected by Flow Cytometry.The transcription levels of α-ENaC mRNA were assessed by qPCR while the protein levels of α-ENaC and p-GSK1 were measured by Western blot.Results 1)Compared with mice in control group, severe pathological lung injury changes were observed in mice of LPS group, with increased total protein levels, PMN proportions,levels of inflammatory cytokines in BALF (P<0.05), accompanied by down-regulated level of α-ENaC and p-SGK1 in lung tissues (P<0.05).2)Compared with mice in LPS group, resveratrol significantly reversed lung injury triggered by LPS, decreased total protein levels, PMN proportions, levels of inflammatory cytokines in BALF (P<0.05), with down-regulated levels of α-ENaC and p-SGK1 in lung tissues (P<0.05).3)However, PP242 prevented beneficial effects of RES on ALI.Conclusions Up-regulation of α-ENaC expression via activation of SGK1 takes part in the protective effects of RES on LPS-induced ALI in mice.

Serum-and glucocorticoid-regulated kinase 1 suppresses inflammatory responses mediated by toll like receptor 4 via nuclear factor-κB / 中华传染病杂志

Objective To investigate the role of serum and glucocorticoid regulated protein kinase (SGK) 1 in the inflammatory responses mediated by toll like receptors.Methods Mice were injected with lipopolysaccharide (LPS,1 mg/kg) 2 h after the pretreatment of EMD638683 (10 mg/kg) or phosphate buffered saline (PBS) as control.At the time points of 3 and 24 h,pro-inflammatory cytokines (interleukin [IL]-6,IL-12 and tumor necrosis factor [TNF]-α) in serum were measured using enzymelinked immunosorbent assay (ELISA).Livers and lung were harvested at 6 h and 24 h after the injection of LPS,embedded by optimum cutting temperature (OCT) and then stained with hematoxylin and eosin (HE).Peripheral blood mononuelear cell (PBMC) were isolated and stimulated by LPS with or without the pretreatment of EMD or LY294002.Cytokines (IL-6,IL-12 and TNF-α) were measured using ELISA.IKKα/β,IKBα and nuclear factor (NF)-κB p65 were detected by Western bolt.Data were analyzed by one way analysis of variance.Results In the model of LPS-induced endotoxin sepsis,inhibition of SGK1 induced secretion of pro-inflammatory cytokine (IL-6 [t=3.007,P＜0.05],IL-12[t=4.413,P＜0.05] and TNF-α[t=5.403,P＜0.05]),increased inflammatory cells infikration into the liver and lung within 6 h,and induced serious multiple organ damage with collapse of alveoli and fatty degeneration of liver.After 24 h,pharmacological inhibition of SGK1 with EMD638683 increased proinflammatory cytokine (IL-6 [t=18.540,P＜0.01],IL-12[t=16.520,P＜0.01] and TNF-α[t=34.880,P＜0.01]) production in human PBMC upon LPS stimulation and inhibited the phosphorylation of IKKα/ β/IKBα and nuclear factor (NF)-κB p65.Conclusions SGK1 suppresses the toll like receptor 4 mediated inflammatory responses via NF-κB.

Effect of SGK1 on the Protection of Ischemia Reperfusion Model Rats / 昆明医科大学学报

Objective To seek the effect of SGK1 on the protection and prognosis of cerebral ischemia reperfusion. Methods Animal model of rats was used to construct them into experimental group(A)and control group(B). The impact of cerebral ischemia reperfusion on the hippocampus neuron cell apoptosis was simulated by making the overexpression of SGK1 and using PI3K inhibitors LY294002 to deal with animal models. Results The overexpression of SGK1 could reduce the neuron cell apoptosis caused by cerebral ischemia reperfusion to a certain extent,reverse the expressions of Cleaved aspas 3 protein,pro-apoptotic protein Bax and inhibitor of apoptoasis protein Bcl-2 through the PI3K/Akt-mediated signaling pathway. Conclusion SGK1 can protect the tissues with ischemia-reperfusion,which may provide the biological evidence for future clinical applications.

Inhibitory effects of eplerenone on cell proliferation via down-regulated SGK-1 pathway in rats with unilateral ureteral obstruction / 中国药理学通报

Aim To observe the effect of mineralocor-ticoid receptor blockade eplerenone on cell proliferation in obstructed kidney of rats. Methods Renal intersti-tial fibrotic animals were made with unilateral ureteral obstruction (UUO) and treated with eplerenone100 mg · kg - 1 · d - 1 . The kidneys were harvested on the 10th day and proliferating cell nuclear antigen ( PC-NA ), serum and glucocorticoid induced kinase-1 (SGK-1 ) and transforming growth factor-β1 ( TGF-β1 ) were detected with immunohistochemistry and Western blot. Results Renal histopathology showed large quantities extracellular matrix (ECM) accumula-tion in kidney with UUO, large numbers of inflammato-ry cells infiltrated in renal interstitium, renal tubular expansion and exfoliation of epithelial cells . The cell proliferation and ECM accumulation were inhibited in eplerenone treated rats significantly. Immunohisto-chemistry and Western blot showed that expressions of PCNA,SGK-1 and TGF-β1 were significantly up-regu-lated with UUO and down-regulated by eplerenone. Conclusion Eplerenone plays the role in inhibiting the cell proliferation and reducing ECM accumulation by down-regulating expression of SGK-1 pathway in rats with unilateral ureteral obstruction.

SGK1-induced imbalance of T lymphocyte subsets in childhood asthma / 国际儿科学杂志

Objective To study the expression of SGK1 in T lymphocytes from pediatric asthma,and the effect of SGK1 on the differentiation of T cells,also to explore the function of SGK1 regulating the differen-tiation of T subset in pediatric asthma. Methods Twenty-eight children with asthma were recruited in Xi′an children′s hospital and divided into moderate group and severe group according to diagnostic guideline of asth-ma. The serum levels of IL-4,IL-13 and IL-17A were analyzed by ELISA. The CD4 +T cells from PBMC and na?ve T cells were selected using magnetic beads. Na?ve T cells were differentiated in vitro under cytokines. SGK1 expression were analyzed with Real-time PCR. The ability of Th2 and Th17 on secreting IL-4 and IL-17A were detected after SGK1 was inhibited by siRNA. In vivo,shRNA-SGK1 Na?ve T cells were transferred into the mice asthma models by intravenous injection. The airway inflammation were observed in shRNA-SGK1 Na?ve T models. Results Compared with healthy children,the serum levels of IL-4、IL-13 and IL-17A increased signifi-cantly in the children with asthma. Importantly,the levels of these three cytokines were much higher with the de-velopment of asthma. SGK1 were up-regulated remarkably in CD4 +T cells from the children with asthma and were positively correlated with IL-13 and IL-17A. Besides,SGK1 expression increased in the differentiated Th2 and Th17 in vitro,but had no change in the differentiated Th1. The levels of IL-4 and IL-17A associated with Th2 and Th17 decreased after SGK1 was inhibited by siRNA. Similarly,In vivo,the serum levels of IL-13 and IL-17A and airway inflammation were reduced in shRNA-SGK1 Na?ve T models. Conclusion The over-expres-sion of SGK1 in pediatric asthma enhances the asthma progress by promoting the differentiation of T subsets.