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ObjectiveTo observe the therapeutic effect of Qiwei Baizhusan（QWBZS） on diabetic encephalopathy（DE） rat model， and to explore the possible mechanism of QWBZS in the treatment of DE based on phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/glycogen synthase kinase-3β（GSK-3β） signaling pathway. MethodForty-eight SPF male Wistar rats were randomly divided into blank group（8 rats） and high-fat diet group（40 rats）. After 12 weeks of feeding， rats in the high-fat diet group were intraperitoneally injected with 35 mg·kg-1 of 1% streptozotocin（STZ） for 2 consecutive days to construct a DE model， and rats in the blank group were injected with the same amount of sodium citrate buffer. After successful modeling， according to blood glucose and body weight， model rats were randomly divided into model group， low， medium and high dose groups of QWBZS（3.15， 6.3， 12.6 g·kg-1）， combined western medicine group（metformin+rosiglitazone， 0.21 g·kg-1）， with 6 rats in each group. The administration group was given the corresponding dose of drug by gavage， and the blank group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage， 1 time/day for 6 weeks. Morris water maze was used to detect the spatial memory ability of DE rats. Fasting insulin （FINS） level was detected by enzyme-linked immunosorbent assay（ELISA） and insulin resistance index（HOMA-IR） was calculated. Hematoxylin-eosin（HE） staining was used to observe the morphological changes of hippocampus in rats， ELISA was used to detect the indexes of oxidative stress in hippocampal tissues， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect mRNA expression levels of PI3K， Akt， nuclear transcription factor-κB（NF-κB）， tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） in hippocampus， and Western blot was used to detect the protein expression of PI3K， Akt， phosphorylated（p）-Akt， GSK-3β and p-GSK-3β in hippocampus of rats. ResultCompared with the blank group， FINS and HOMA-IR values of the model group were significantly increased（P<0.01）， the path of finding the original position of the platform was significantly increased， and the escape latency was significantly prolonged（P<0.01）， the morphology of neuronal cells in hippocampal tissues was disrupted， the levels of reactive oxygen species（ROS） and malondialdehyde（MDA） in hippocampus of rats were increased， and the activity of superoxide dismutase（SOD） was decreased（P<0.05， P<0.01）， mRNA expression levels of PI3K and Akt were decreased（P<0.01）， mRNA expression levels of NF-κB， TNF-α and IL-1β were increased（P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly decreased， and the protein expression of GSK-3β was significantly increased（P<0.01）. Compared with the model group， the FINS and HOMA-IR values of the medium dose group of QWBZS and the combined western medicine group were significantly decreased（P<0.01）， the path of finding the original position of the platform and the escape latency were significantly shortened（P<0.01）， the hippocampal tissue structure of rats was gradually recovered， and the morphological damage of nerve cells was significantly improved， the contents of ROS and MDA in hippocampus of rats decreased and the level of SOD increased（P<0.01）， the mRNA expression levels of PI3K and Akt were increased（P<0.01）， and the mRNA expression levels of NF-κB， TNF-α and IL-1β were decreased （P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly increased（P<0.01）， and the expression of GSK-3β was significantly decreased（P<0.01）. ConclusionQWBZS can alleviate insulin resistance in DE rats， it may repair hippocampal neuronal damage and improve learning and cognitive ability of DE rats by activating PI3K/Akt/GSK-3β signaling pathway.
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AIM: To investigate the PI3K/AKT/GSK-3β signaling pathway involved in the protective effect and mechanism of propofol on the cerebral ischemia-reperfusion injury in rats. METHODS: There were 72 healthy male SD rats. All rats established a model of focal cerebral ischemia-reperfusion injury according to the Zea Longa method and were randomly divided into six groups (n=12), A-sham operation group, B-model group (MCAO), C-Propofol group, D-Propofol+adenosine A1R antagonist group (DPCPX), E-Propofol group+PI3K specific inhibitor (LY294002), F-Propofol+GSK3β inhibitor group (SB216763). The neurological scores of rats 24 h after operation, LDF monitors changes in cerebral blood flow before and after embolization were observed. The TTC staining method was used to detect the cerebral infarction volume of rats in each group; HE staining method was used to observe the morphological changes of the rat brain tissue; Immunohistochemical method was used to detect Bcl-2 positive cells expression; TUNEL was used to detect cerebral cortex ischemia in each group. The percentage of neuronal apoptotic cells. RESULTS: Compared with group A, the behaviors, cerebral infarction volume, apoptosis rate, and Bcl-2 protein expression of rats in groups B, C, D, E, and F all increased (P<0.05); compared with group C, the behavioral scores, cerebral infarction volume and apoptosis rate of rats in groups B, D and E all increased significantly, and the expression of Bcl-2 protein was decreased significantly (P<0.01), but the expression of Bcl-2 protein in group F was increased, cell apoptosis rate decreased (P<0.05), behavior score and infarcts decreased (P<0.05). CONCLUSION: The neuroprotective effect of propofol mediated by adenosine A1R on ischemia-reperfusion injury in rats may be related to the PI3K/AKT/GSK-3β signal transduction pathway.
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Objective To observe the protective effect of hydroxysafflor yellow A(HSYA) on anoxia/reoxygenation (A/R) injury of neonatal primary cardiomyocytes, and its relationship with phosphoinositide 3-kinase/protein ki-nase B/glycogen synthase kinase 3β(PI3K/Akt/GSK3β) signaling pathway. Methods Primary cardiomyocytes of neonatal rats were isolated from the rats and incubated for 48 hours. The cells were adhered to each other and then divided into five groups:control group (Con group), anoxia/reoxygenation group (A/R group),HSYA treatment group(A/R+H group),PI3K inhibitor (LY294002)treatment group(A/R+L group)and HSYA+LY294002 treat-ment group (A/R+H+L group),then to collect the supernatant fluid of each group to measure LDH.The flow cy-tometry was used to measure the apoptotic cells. The protein levels of Bcl-2,Bax,Akt,p-Akt (Ser473),GSK3β, p-GSK3β (Ser9) were evalated by Western blot. Results A/R increased LDH release,the apoptosis rate (P<0.001),and the expression of pro-apoptotic protein Bax (P <0.001) with the decrease of anti-apoptotic protein Bcl-2,p-Akt(Ser473), p-GSK3β(Ser9)(P<0.001) as compared with the control group. HSYA treatment de-creased LDH release,the apoptosis rate (P<0.001),and the expression of Bax (P<0.001) and increase the ex-pression of Bcl-2,p-Akt(Ser473),p-GSK3β(Ser9)(P<0.001). Compared with the A/R+H group,the expres-sion of Bax was increased (P<0.001),while the expression of Bcl-2, p-Akt(Ser473), p-GSK3β(Ser9)was de-creased (P<0.001) in the A/R+H+L group. Conclusions HSYA protects rats'cardiomyocytes from anoxia/reoxy-genation injury by regulating PI3K/Akt/GSK3β signaling pathway.
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Aim To investigate the effect of Ginsen-oside Rh2 on apoptosis in human colorectal cancer cell SW480,and to explore the possible mechanism of it. Methods The proliferation activity of SW480 treated with Ginsenoside Rh2 was measured CCK-8 assay.Ap-optosis rates were evaluated by FCM.Hoechst 33258 staining was used to observe cell nucleus morphologi-cal;change SW480 cells were treated with Ginsenoside Rh2,and the protein expressions of Bcl-2,Bax,p53, cleaved caspase-3 ,PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot;SW480 cells were treated with LY294002,Rh2,LY294002+Rh2, the expressions of PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot.Results The proliferation of SW480 cells was significantly inhibited by Ginsenoside Rh2 in dose-dependent and time-de-pendent manner.FCM showed the inducing apoptosis effect of Ginsenoside Rh2 was significantly different from that of control group.Hoechst 33258 staining in-dicated clearly cell apoptosis in Ginsenoside Rh2 treat-ment groups.Western blot showed Ginsenoside Rh2 decreased expression of Bcl-2,increased expression of Bax,p53 and cleaved caspase-3,PI3K/AKT/GSK-3βpathway proteins PI3 K,P-AKT,P-GSK-3βdecreased obviously,AKT and GSK-3βwere not changed signifi-cantly in SW480.SW480 cells were separately treated with LY294002,Rh2,LY294002 +Rh2,there were no significant difference in AKT and GSK-3βprotein a-mong all groups,and the expression of PI3 K,P-AKT, P-GSK-3βdecreased more obviously in LY294002 +Rh2 group compared with LY294002 and Rh2 alone. Conclusion Rh2 induces colorectal cancer cell apop-tosis through PI3 K/AKT/GSK-3βpathway,which ac-tivates p53 and cleaved caspase-3,and destroys the balance of Bcl-2/Bax.
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Objective To study the changes of PI3K/Akt/GSK3β signaling pathway during resuscitation with neck cooling in order to explore the relationship between the protective effect of neck cooling and the phosphorylation of PI3K/Akt and GSK3β.Methods Thirty rabbits were randomly(random number) divided into five groups, and models of cadiac arrest were induced by ventricular fibrillation(VF, the positive electrode in the right ventricle and negative pole on the apex of heart) for 4 min.In sham group,a electrode was placed into right ventricle without electric current conducted, and CA was not induced.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.In normothermia treat group(NT group),resuscitation was carried out to restoration of spontaneous circulation(ROSC),and the rabbits were sacrificed and specimens were taken at 24 hours after modeling.In intra-arrest therapeutic hypothermia group (IATH group), rapid neck cooling was initiated at the same time with CPR,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.Rabbits were sacrificed and specimens were taken at 24 hours after modeling.In recovery period cooling + LY294002 group(PATH+LY294002 group), LY294002 was injected intra-ventricularly at 20 minutes before resuscitation.Rapid neck cooling was started at the same time with CPR,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.In post-arrest therapeutic hypothermia group (PATH group), rapid neck cooling was begun after CPR for 1 hour,and the target brain temperature was set at 34 ℃ maintained for 4 hours after ROSC.The rabbits were sacrificed and specimens were taken at 24 hours after modeling.Animals were sacrificed by using overdose anesthetic drug.Western blot was used to detect the level of Akt p-Akt GSK-3β p-GSK-3β (ser9) protein, and TUNEL was used to observe apoptosis of tissues in each group.Multiple comparisons were performed with one-way analysis of variance (ANOVA).Results Compared with Sham group, Akt (Thr-308) phosphorylation (P-AKT) and P-GSK-3β levels in the brain neuron cytoplasm in 24 hours after CPR resuscitation in NT group was significantly reduced, and showed a gradual reduction trend (P<0.05);the P-AKT and P-GSK-3β levels in the brain neuron cytoplasm in 24 hours after CPR resuscitation in IATH group were significantly enhanced compared with NT group (P<0.05);the levels of these two kinds of protein at one hour after resuscitation in PATH group were significantly enhanced compared with NT group (P<0.05), but lower in IATH group.Intra-ventricularly injection of LY294002 made the effect of hypothermia lost, indicating that LY294002 inhibited the phosphorylation of Akt.Apoptosis cells were significantly reduced in IATH group and normothermia theatment group compared with PATH group and LY294002 group(P<0.05).Conclusions Neck cooling can reduce apoptosis in rabbit brain cells after recovery, and the protective effect on brain is best in intra-arrest therapeutic hypothermia group.LY294002 specifically block the PI3K/Akt pathway, and the protective effect of cooling on the brain can be abolished,indicating hypothermia protects the neurological function via activation of PI3K/Akt pathway.Neck cooling protects the neurological function by activating PI3K/Akt/GSK-3β, promoting the Akt activation, and increasing the expression of P-GSK3β.Specific Akt inhibitor LY294002 inhibits Akt phosphorylation of brain tissue recovery and further inhibit the phosphorylation of GSK-3β, thus abolishing protective effect of cooling on neurological function.