ABSTRACT
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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Objective:To investigate the effect of baicalin on cognitive function of mice with brain injury induced by mechanical ventilation and its mechanism.Methods:Seventy two C57BL6 mice, weighing 20-25 g, aged 8-12 weeks, were randomly divided into control group (group C), mechanical ventilation group (group V), baicalin group (group B), baicalin+ Akt inhibitor MK-2206 group (group BM) according to random number table method, with 18 in each group.Mice in group C did not have mechanical ventilation and breathed air independently for 6 hours.Mice in group V received mechanical ventilation for 6 hours.Mice in group B and group BM were intraperitoneally injected with baicalin 100 mg/kg 30 minutes before mechanical ventilation, and mice in group BM were injected intraventricular with Akt inhibitor MK-2206 300 μg/kg 60 minutes before mechanical ventilation.Six mice in each group were randomly selected to test their learning and memory abilities by Morris water maze test 1st day before mechanical ventilation and 3rd day and 7th day after mechanical ventilation.One day after mechanical ventilation, six mice in each group were killed, and the brain tissue was taken.TUNEL method was used to detect the neuronal apoptosis in hippocampal CA1 area, and the apoptosis index was calculated.One day after mechanical ventilation, six mice in each group were killed, and the hippocampus was taken, Western blot was used to detect the protein expressions of caspase-3, caspase-9, Akt, p-Akt, GSK-3β and p-GSK-3β.SPSS 22.0 software was used for statistical analysis of data, repeated measure ANOVA and one-way ANOVA were used for comparison between multiple groups.LSD- t test was used for further pairwise comparison. Results:The results of water maze test showed that the time and group interaction of the four groups were not significant ( F=1.14, P>0.05), the main effect of time and group were both significant ( F=47.36, 59.65, both P<0.05). At 3rd day and 7th day after mechanical ventilation, the escape latencies of mice in group V were higher than those in group C (both P<0.05), and the numbers of platform crossing were lower than those in group C (both P<0.05). And 3 days and 7 days after mechanical ventilation, the escape latencies of mice in group B were lower than those in group V (both P<0.05) and the numbers of platform crossing were higher than those in group V (both P<0.05). The escape latenies of mice in BM group on the 3rd and 7th day were higher than those in group B (both P<0.05), and the numbers of platform crossing were lower than those in group B on the 3rd day and 7th day after mechanical ventilation(both P<0.05). TUNEL and Western blot results showed that apoptosis index of hippocampal neurons and expression levels of apoptosis-related proteins caspase-3 and caspase-9 were significant different in the four groups ( F=51.42, 41.21, 40.19, all P<0.05). The apoptosis index of hippocampal neurons ((40.6±3.9)%), the expression levels of caspase-3 (4.93±0.92) and caspase-9 (4.81±0.88) in the hippocampus of mice in group V were higher than those in group C ((13.7±1.4)%, (1.87±0.27), (1.71±0.25), all P<0.05), the apoptosis index of hippocampal neurons ((15.6±1.6)%), the expression levels of caspase-3 (1.95±0.30) and caspase-9 (1.76±0.28) in group B were lower than those in group V ((40.6±3.9)%, (4.93±0.92), (4.81±0.88), all P<0.05), the apoptosis index of hippocampal neurons ((27.8±2.7)%), the expression levels of caspase-3 (3.58±0.61) and caspase-9 (3.49±0.57) in BM group were higher than those in group B ((15.6±1.6)%, (1.95±0.30), (1.76±0.28), all P<0.05). Expression level of p-Akt, p-GSK-3β in hippocampal tissues of the four group of mice were significantly different ( F=37.54, 43.23, both P<0.05). The expression level of p-Akt (0.51±0.06) and p-GSK-3β (0.47±0.05) of hippocampal tissues of mice in group V were lower than those of group C ((1.07±0.10), (1.11±0.12), both P<0.05), the expression level of p-Akt (0.99±0.10) and p-GSK-3β (1.08±0.09) of hippocampal tissues of mice in group B were higher than those of group V (both P<0.05), the expression level of p-Akt (0.83±0.08) and p-GSK-3β (0.81±0.07) of hippocampal tissues of mice in group BM were lower than those in group B (both P<0.05). Conclusion:Baicalin can improve the cognitive function of mice with brain injury induced by mechanical ventilation, which is related with activation of Akt/GSK-3β signaling pathway and inhibition of hippocampal neuron apoptosis.
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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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OBJECTIVE: To investigate the effect of ABT-263,an anti-apoptotic protein inhibitor,on human cutaneous squamous cell carcinoma A431 cells,and to explore its molecular mechanisms. METHODS: i) Total protein was extracted from human immortalized epidermal cells( Ha Ca T cells) and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2( BCL-2) and BCL2-like 1( BCL-XL) was detected by Western blotting. ii) The A431 cells were treated with ABT-263( inhibitor group) and dimethyl sulfoxide( control group) at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy. iii) The A431 cells were treated with 0,10,25,40,and 50 μmol/L of ABT-263 for 24 hours,and the cell viability was determined by CCK-8 assay. iv) The A431 cells were treated with different doses of ABT-263,and the expression of cleaved Caspase-3, cleaved poly( ADP-ribose) polymerase-1( PARP-1), phosphorylated protein kinase B [p AKT(ser473)],phosphorylated glycogen synthase kinase-3β(p GSK3β) and phosphorylated histone H2 AX(γH2 AX) was detected by Western blot. RESULTS: The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in Ha Ca T cells( P < 0. 01). Transmission electron microscopy results showed that A431 cells in inhibitor group gradually changed from normal morphology to apoptotic morphology,showing loss of microvilli,increased nuclear chromatin density and aggregation around the nuclear membrane,and nuclear fragmentation. The cell viability of A431 cells in 10,25,40 and 50 μmol/L groups were lower than those in control group( P < 0. 05). The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10,30 and 50 μmol/L groups were higher than those in control group( P < 0. 05).The relative expression of p AKT( ser473) and p GSK3β in A431 cells in 10,25,40 and 50 μmol/L groups were lower than those of the control group( P < 0. 05) and γH2 AX protein expression was higher than that of the control group( P <0. 05). A431 cell viability and p GSK3β protein expression decreased with the increase of inhibitor dosage( P < 0. 01).The relative expression of cleaved Caspase-3 and γH2 AX protein increased with the increase of inhibitor dosage( P <0. 01),showing dose-effect relationship. CONCLUSION: ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3β pathway,which can promote the apoptosis of A431 cells with a doseeffect relationship.