Intervention Mechanism of Biejiajian Wan on Primary Liver Cancer by Regulating lncRNA SNHG5/miRNA-26a-5p/GSK-3β Signal Axis / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.

Effect of Danzhi Xiaoyaosan on Phosphorylation of Tau Protein in Rats with Alzheimer's Disease Based on PP2A and GSK-3β / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Danzhi Xiaoyaosan on the phosphorylation of tau protein and different sites of glycogen synthase kinase-3β （GSK-3β） and phosphoseryl/suanyl phosphate protein phosphatase 2A （PP2A） in the hippocampus of rats with Alzheimer's disease （AD） and its mechanism. MethodThe rat model of AD was established by injecting okadaic acid into the bilateral hippocampus of 90 male Wistar rats in SPF grades. The rats with successful modeling were selected and randomly divided into model group， aricept group （0.5 mg·kg-1）， and Danzhi Xiaoyaosan high， medium， and low groups （17.55， 8.77， and 4.38 g·kg-1）， and then gavaged for 42 d， once a day. Morris water maze was used to detect the learning and memory ability of rats， Nissl's staining was used to observe the morphological structure of neurons in the hippocampus， and Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tau protein， GSK-3β， and PP2A. Western blot was used to determine the protein expression levels of tau protein， GSK-3β， and PP2A. ResultAs compared with the control group， the learning and memory abilities of the rats in the model group were significantly decreased （P<0.01）， and the hippocampal CA3 region cells had abnormal structure， disorderly arrangement， and decreased number. The expression levels of GSK-3β mRNA， GSK-3β， p-GSK-3β-Tyr216， p-PP2A， and p-tau were increased in the model group as compared with the control group （P<0.01）， and those of p-GSK-3β-Ser9 and PP2A decreased significantly （P<0.01）. As compared with the model group， the learning and memory ability of the Aricept group and the Danzhi Xiaoyaosan groups were improved （P<0.05， P<0.01）， and the cell morphology and the number of hippocampal CA3 regions were better. The mRNA expression levels of PP2A and tau in the Aricept group were significantly up-regulated （P<0.05）， the mRNA expression level of GSK-3β was significantly down-regulated （P<0.01）， and the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-PP2A were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A in the high-dose Danzhi Xiaoyaosan group was significantly up-regulated （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of p-PP2A， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of GSK-3β was significantly down-regulated in the medium-dose Danzhi Xiaoyaosan group （P<0.01）， the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A was significantly up-regulated in the low-dose Danzhi Xiaoyaosan group （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of GSK-3β and p-GSK-3β-Tyr216 were down-regulated （P<0.05， P<0.01）， and those of p-GSK-3β-Ser9 and PP2A were significantly up-regulated （P<0.01）. ConclusionDanzhi Xiaoyaosan can improve the learning and memory ability of rats with AD， and its mechanism may be related to the regulation of the activities of GSK-3β and PP2A protein-related sites and the phosphorylation of tau protein.

Effects of Yizhi Tiaoshen acupuncture on learning and memory function and the expression of phosphorylated tau protein in the hippocampus of Alzheimer's disease model rats / 中国针灸

OBJECTIVE@#To observe the effects of Yizhi Tiaoshen (benefiting mental health and regulating the spirit) acupuncture on learning and memory function, and the expression of phosphorylated tubulin-associated unit (tau) protein in the hippocampus of Alzheimer's disease (AD) model rats, and explore the effect mechanism of this therapy on AD.@*METHODS@#A blank group and a sham-operation group were randomly selected from 60 male SD rats, 10 rats in each one. AD models were established in the rest 40 rats by the intraperitoneal injection of D-galactose and okadaic acid in the CA1 region of the bilateral hippocampus. Thirty successfully-replicated model rats were randomly divided into a model group, a western medication group and an acupuncture group, 10 rats in each one. In the acupuncture group, acupuncture was applied to "Baihui" (GV 20), "Sishencong" (EX-HN 1), "Neiguan" (PC 6), "Shenmen" (HT 7), "Xuanzhong" (GB 39) and "Sanyinjiao" (SP 6); and the needles were retained for 10 min. Acupuncture was given once daily. One course of treatment was composed of 6 days, with the interval of 1 day; the completion of treatment included 4 courses. In the western medication group, donepezil hydrochloride solution (0.45 mg/kg) was administrated intragastrically, once daily; it took 7 days to accomplish one course of treatment and a completion of intervention was composed of 4 courses. Morris water maze (MWM) and novel object recognition test (NORT) were used to assess the learning and memory function of the rats. Using HE staining and Nissl staining, the morphological structure of the hippocampus was observed. With Western blot adopted, the protein expression of the tau, phosphorylated tau protein at Ser198 (p-tau Ser198), protein phosphatase 2A (PP2A) and glycogen synthase kinase-3β (GSK-3β) in the hippocampus was detected.@*RESULTS@#There were no statistical differences in all of the indexes between the sham-operation group and the blank group. Compared with the sham-operation group, in the model group, the MWM escape latency was prolonged (P<0.05), the crossing frequency and the quadrant stay time in original platform were shortened (P<0.05), and the NORT discrimination index (DI) was reduced (P<0.05); the hippocampal cell numbers were declined and the cells arranged irregularly, the hippocampal neuronal structure was abnormal and the numbers of Nissl bodies decreased; the protein expression of p-tau Ser198 and GSK-3βwas increased (P<0.05) and that of PP2A decreased (P<0.05). When compared with the model group, in the western medication group and the acupuncture group, the MWM escape latency was shortened (P<0.05), the crossing frequency and the quadrant stay time in original platform were increased (P<0.05), and DI got higher (P<0.05); the hippocampal cell numbers were elevated and the cells arranged regularly, the damage of hippocampal neuronal structure was attenuated and the numbers of Nissl bodies were increased; the protein expression of p-tau Ser198 and GSK-3β was reduced (P<0.05) and that of PP2A was increased (P<0.05). There were no statistically significant differences in the above indexes between the acupuncture group and the western medication group (P>0.05).@*CONCLUSION@#Acupuncture therapy of "benefiting mental health and regulating the spirit" could improve the learning and memory function and alleviate neuronal injure of AD model rats. The effect mechanism of this therapy may be related to the down-regulation of GSK-3β and the up-regulation of PP2A in the hippocampus, and then to inducing the inhibition of tau protein phosphorylation.

Mechanism of Modified Shenqiwan in Relieving Renal Interstitial Fibrosis in Diabetic Mice Based on GSK-3β/CREB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of modified Shenqiwan on renal function and fibrosis in diabetic nephropathy mice and explore the underlying mechanism based on the glycogen synthase kinase-3β （GSK-3β）/cyclic adenosine monophosphate （cAMP） response element-binding protein （CREB） signaling pathway. MethodFifty male db/db mice and 10 db/m mice were used in this study. The fifty db/db mice were randomly divided into model group， irbesartan group， and low-， medium-， and high-dose modified Shenqiwan groups. The 10 db/m mice were assigned to the normal group. The mice in the low-， medium-， and high-dose modified Shenqiwan groups were administered with modified Shenqiwan in the dosage form of suspension of Chinese medicinal granules by gavage， those in the irbesartan group were given irbesartan suspension by gavage， and those in the normal and model groups were given distilled water of equal volume by gavage. The intervention lasted for 12 weeks. The blood glucose levels， urine albumin-to-creatinine ratio （UACR）， and the protein expression levels of GSK-3β， CREB， transforming growth factor-β1 （TGF-β1）， E-cadherin， Vimentin， fibronectin （FN）， plasminogen activator inhibitor-1 （PAI-1）， and Collagen type Ⅳ （Coll Ⅳ） in the mouse kidneys were recorded before and after treatment. The extent of renal pathological damage was also observed. ResultCompared with the normal group， the model group showed significant increases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， decreased protein expression level of CREB （P<0.05）， and severe renal pathological damage. Compared with the model group， the low-， medium-， and high-dose modified Shenqiwan groups and the irbesartan group showed varying degrees of decreases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， increased expression level of CREB protein （P<0.05）， and improved renal pathological damage. ConclusionModified Shenqiwan can effectively reduce blood glucose levels， improve renal function， and alleviate fibrosis， and the mechanism of action is related to the inhibition of the GSK-3β/CREB signaling pathway.

Icariin Inhibits Proliferation of CLC5 Hepatoma Cells via Akt/GSK3β/CDK Pathway / 中国实验方剂学杂志

ObjectiveTo study the effect of icariin on the proliferative capacity of hepatocellular carcinoma cell line CLC5 and the underlying mechanism. MethodThe targets of icariin were screened out by network pharmacology， and the target network and protein-protein interaction （PPI） network were constructed to predict the possible targets and pathways of icariin. CCK-8 assay was employed to explore the effects of different concentrations （0， 6.25， 12.5， 25， 50 μmol·L-1） of icariin on the viability of CLC5 cells. Further， CLC5 cells were treated with 0， 25， 50 μmol·L-1 icariin， and the effect of icariin on CLC5 cell proliferation was examined by Edu-488 assay and clone formation assay （CFA）. Western blot was employed to measure the expression levels of proteins in the protein kinase B （Akt）/glycogen synthase kinase 3β （GSK3β）/cell cycle-dependent kinase （CDK） pathway in the CLC5 cells exposed to different concentrations of icariin. ResultNetwork pharmacological analysis revealed that icariin may inhibit the hepatocellular carcinoma via cell cycle arrest and inhibition of tumor cell proliferation. Compared with the blank group， icariin decreased the viability of CLC5 cells in a time- and concentration-dependent manner （P<0.01） and reduced the positive rate of Edu-488 and the colonies in CFA （P<0.05， P<0.01）. Moreover， icariin down-regulated the protein levels of p-Akt， p-GSK3β， CDK4， and CyclinD1 （P<0.05， P<0.01）. ConclusionIcariin may block cell cycle to suppress the proliferation of CLC5 cells via inhibiting the Akt/GSK3β/CDK pathway.

Effect of Zuoguiwan on Wnt/β-catenin Signaling Pathway in Ovariectomized Osteoporosis Model Rats / 中国实验方剂学杂志

Objective:To observe the effect of Zuoguiwan on bone metabolism and Wnt/<italic>β</italic>-catenin signaling pathway in ovariectomized osteoporotic rats model， and to explore the molecular biological mechanism of Zuoguiwan in the prevention and treatment of osteoporosis. Method:The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy， 60 female SD rats were randomly divided into sham operation group， model group， positive group （estradiol valerate tablet 0.05 mg·kg^-1·d^-1） and low， middle and high dose groups of Zuoguiwan （5.5，11，22 g·kg^-1·d^-1）.After successful establishment of the model in the 13^th week， intragastric administration （<italic>ig</italic>） was given once a day for a total of 12 weeks. After administration， the histomorphological changes of femur in rats were observed by hematoxylin-eosin （HE） staining， the bone mineral density （BMD） and bone mineral content（BMC） of femur were measured by dual energy X-ray apparatus， and the biomechanical properties of bone were measured by MTS Acumen3 biomechanical testing system. The contents of bone alkaline phosphatase （BALP）， bone glaprotein（BGP），estradiol （E₂） ，and tartrate-resistant acid phosphatase （TRAP）， type Ⅰ procollagen N-terminal propeptide （PINP） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the protein level of Wnt2，<italic>β</italic>-catenin，low density lipoprotein related receptor protein 5 （LRP5） and the phosphorylation level of glycogen synthase kinase-3<italic>β</italic>（GSK-3<italic>β</italic>） in rat tibia. Result:Compared with sham operation group， the maximum load and stiffness of BMD，BMC， in the model group decreased significantly（<italic>P</italic><0.01）， the contents of E₂ and PINP in serum decreased significantly（<italic>P</italic><0.01）， the content of BALP，BGP，TRAP increased significantly（<italic>P</italic><0.01）， the expression levels of Wnt2，p-GSK-3<italic>β </italic>Ser9，LRP5 and <italic>β</italic>-catenin protein in bone tissue decreased significantly（<italic>P</italic><0.01）， the trabecula of femur became thinner and thinner， the number of bone trabeculae decreased. Compared with model group， the maximum load and stiffness of BMD，BMC， in estradiol group and Zuoguiwan group were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the contents of serum E₂ and PINP were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the content of BALP，BGP，TRAP was significantly decreased （<italic>P</italic><0.01）， and the expression level of Wnt2，p-GSK-3<italic>β</italic> Ser9，LRP5， <italic>β</italic>-catenin protein in bone tissue was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01） ， the trabeculae of femur became thicker， the number increased， the structure was basically clear. Conclusion:Zuoguiwan has a certain preventive and therapeutic effect on osteoporosis in ovariectomized rats， and its mechanism may be related to increasing the level of estrogen， activating Wnt/<italic>β</italic>-catenin signaling pathway， up-regulating the expression of Wnt2 and LRP5 protein， inhibiting the activity of GSK-3<italic>β</italic>， reducing the degradation of <italic>β</italic>-catenin， coordinating the dynamic coupling balance between bone formation and bone resorption， correcting the disorder of bone metabolism and improving bone morphology.

Effect of Huazhuo Jiedu Shugan Prescription on Learning， Memory， and PI3K/Akt/GSK-3β Signaling Pathway in Epileptic Rats / 中国实验方剂学杂志

Objective:To observe the effects of Huazhuo Jiedu Shugan Prescription （HZJDSG） on learning， memory， and the expression of phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt）/glycogen synthase kinase-3<italic>β</italic> （GSK-3<italic>β</italic>） pathway-related proteins in epileptic rats， and to explore its possible mechanism. Method:Forty-eight SPF male SD rats were randomly divided into a normal group， a model group， a sodium valproate （0.19 g·kg^-1） group， and low- （2.7 g·kg^-1）， medium- （5.4 g·kg^-1）， and high-dose （10.8 g·kg^-1） HZJDSG groups， with eight rats in each group. The normal group received 0.9% sodium chloride solution （0.035 g·kg^-1） by intraperitoneal injection， and the other five groups received pentetrazol （PTZ） at the same dose to induce a chronic epilepsy model for a total of 14 times. The drug groups received corresponding drugs and the normal group and the model group received 0.9% sodium chloride solution at the same volume once a day for 28 days. During the drug intervention period， epilepsy was maintained in each modeling group by intraperitoneal injection of PTZ on day 7， 14， 21， and 28. The behavioral changes of rats were observed by Morris water maze and the pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin （HE） staining. The protein expression of phosphorylation Akt（p-Akt）and p-GSK-3<italic>β</italic> was detected by immunohistochemistry and the protein expression of PI3K， Akt， p-Akt， GSK-3<italic>β</italic>， and p-GSK-3<italic>β</italic> by Western blot. Result:Compared with the normal group， the model group showed prolonged platform finding time （<italic>P</italic><0.01）， reduced number of platform crossings （<italic>P</italic><0.01）， structural damage of neurons in the CA1 region of the hippocampus， down-regulated protein expression of p-Akt and p-GSK-3<italic>β </italic>in the CA1 region of the hippocampus （<italic>P</italic><0.05）， and reduced relative expression of PI3K， p-Akt， and p-GSK-3<italic>β</italic> in the hippocampus （<italic>P</italic><0.01）. Compared with the model group， the sodium valproate group and the HZJDSG groups showed shortened platform finding time （<italic>P</italic><0.01） and improved neuronal structure in the CA1 region of the hippocampus， while the sodium valproate group and the high- and medium-dose HZJDSG groups exhibited increased number of platform crossings （<italic>P</italic><0.01）， up-regulated protein expression of p-Akt and p-GSK-3<italic>β</italic> in the CA1 region of the hippocampus （<italic>P</italic><0.05）， and elevated relative expression of PI3K， p-Akt， and p-GSK-3<italic>β</italic> （<italic>P</italic><0.01）. Conclusion:HZJDSG can improve the learning and memory of epileptic rats， and its antiepileptic effect may be achieved by the activation of PI3K/Akt/GSK-3<italic>β</italic> pathway-related proteins.

Effect of TSG on GSK3β，PKA and PP2A of APP/PS1/Tau Triple-transgenic Mice Dementia Model / 中国实验方剂学杂志

Objective:To observe the effect of tetrahydroxy stilbene glycoside （TSG） on the expression of glycogen synthase kinase 3<italic>β </italic>（GSK3<italic>β</italic>）， cyclic adenosine monophosphate-dependent protein kinase （PKA） and Serine/threonine phosphatase 2A（PP2A） in the brain of amyloid precursor protein/presenilin-1/Tau （APP/PS1/Tau） triple-transgenic mice dementia model. Method:A total of forty-five 8-month-old APP/PS1/Tau transgenic mice were randomly divided into model group， positive control group （Huperzine-A， 0.15 mg·kg^-1）， low， medium and high dose TSG groups （TSG， 0.033，0.1，0.3 g·kg^-1）， with 9 mice in each group， and another nine C5B7L/6J mice of the same age were selected as normal control group. After 60 days of intragastric administration， the general structure of hippocampal neurons was observed by hematoxylin-eosin （HE） staining， immunohistochemical （IHC） was used to detect the expression of PKA protein in the brain of mice in each group， the mRNA expression levels of GSK3<italic>β</italic>， PKA and PP2A were detected by real time quantitative reverse transcription polymerase chain reaction （Real-time PCR）， and protein expression levels of GSK3<italic>β</italic> and PP2A were detected by Western blot. Result:Compared with the normal control group， the apoptosis level of neurons in the model group was significantly increased， the protein and mRNA expression levels of GSK3<italic>β</italic> and PKA were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， and the protein and mRNA expression levels of PP2A were significantly decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the apoptosis level of neurons in each treatment group was significantly down-regulated， the protein and mRNA expression levels of GSK3<italic>β</italic> and PKA were significantly down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， and the protein and mRNA expression levels of PP2A were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:The mechanism of TSG in the treatment of Alzheimer's disease （AD） may be related to lowering the transcription and expression of GSK3<italic>β</italic> and PKA， increasing the transcription and expression of PP2A.

Mechanism of Huangjingwan in Inhibiting Tau Hyperphosphorylation in Hippocampal Neurons of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid Resulting in Learning and Memory Disorders / 中国实验方剂学杂志

Objective:To investigate the effect of Huangjingwan （HW） on the activities of glycogen synthase kinase-3β （GSK-3β）， protein phosphatase 2A （PP2A） and the mechanism in inhibiting tau protein hyperphosphorylation in the hippocampal neurons of mice with Alzheimer's disease. Method:After subcutaneous injection with 1.0% D-galactose （0.14 g·kg-1·d-1） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL （75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10-3 g·kg-1·d-1） and HW group （2.5 g·kg-1·d-1）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group for modeling control. Two weeks after modeling， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. The mice in normal control group were only given daily feed. At the end of gavage， all the mice were tested by the open field experiment and jumping platform experiment to evaluate the differences in exploratory activity ability， anxiety level and learning and memory ability. The number of neurons in CA1 and CA3 areas of hippocampus in all the mice was detected by Nissl staining. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to detect mRNA expressions of GSK-3β and PP2A in hippocampus of mice in each group. Protein expressions of GSK-3β， PP2A， phosphorylated tau （p-tau） and total tau protein （t-tau） in hippocampus of mice in each group were detected by Western blot. Result:Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by a lower spontaneous activity， lower exploration behavior ability， higher anxiety level， less movement and easier to stay and hide， longer learning response time， significantly increased number of learning and memory errors， and decreased numbers of hippocampal neuron in CA1 and CA3 areas， and reduced mRNA and protein expressions of PP2A， mRNA and protein expressions of GSK-3β， p-tau protein and the ratio of p-tau/t-tau were all increased significantly （P<0.01）， while expression of t-tau protein was decreased， with no significant difference. Compared with the AD model group， mice in the HW group showed a higher spontaneous activity， higher exploration ability， lower anxiety level， higher learning and memory performance， and the numbers of hippocampal neuron in CA1 and CA3 areas increased， while mRNA and protein expressions of PP2A increased， and the mRNA and protein expressions of GSK-3β， the expression of p-tau protein and the ratio of p-tau/t-tau were all decreased significantly （P<0.01）， but with no significant difference in the protein expression of t-tau. Conclusion:HW can inhibit tau hyperphosphorylation in hippocampal neurons of AD mice， restore tau protein function， protect hippocampal neurons， and exert an anti-AD effect， which may be related to the regulatory mechanism in the activity balance between GSK-3β and PP2A in hippocampal neurons.

Lung Protective Mechanism of Compound Kushen Injection on Radiation-induced Pulmonary Injury / 中国实验方剂学杂志

Objective::To observe the effect of compound Kushen injection on the expressions of transforming growth factor-β1 (TGF-β1), drosophila mothers against decapentaplegic protein 3 (Smad3), glycogen synthase kinase-3β (GSK-3β) and β-catenin mice models with radiation-induced pulmonary injury (RIPI), in order to explore its possible mechanism of action. Method::On XStrahl precision radiation research platform for small animals (SARRP), a single 20 Gy bilateral lung field irradiation was performed to establish a mice model of RIPI. Thirty-two mice were randomly divided into normal control group, model group, compound Kushen injection group and dexamethasone injection group. The normal control group and the model group were given an equal volume of 0.9%sodium chloride solution and injected intraperitoneally for 4 weeks. The pathology of lung tissue tissues was observed by using hematoxylin-eosin (HE) staining. Immunohistochemical(IHC) was used to detect the expressions of E-cadheren and Vimentin proteins in mice lung tissues.Real-time polymerase chain reaction (Real-time PCR) was used to detect the mRNA expressions of TGF-β1, Smad3, GSK-3β and β-cateninin.Western blot was used to detect the protein expressions of TGF-β1, Smad3, GSK-3β and β-cateninin. Result::Compared with the normal group, the pulmonary coefficient of the model group was significantly decreased (P<0.01). Inflammatory cell infiltration, pulmonary interstitial edema, congestion, destruction of alveolar structure and partial alveolar atrophy were observed in the lung tissues of the model group. Compared with the model group, in the compound Kushen injection group, the levels of infiltration of lung inflammatory cells and pulmonary interstitial lesions in mice, the expression of Vimentin in lung tissues (P<0.01), and the expressions of TGF-β1, Smad3, GSK-3β and β-cateninin were significantly decreased (P<0.01), whereas the expression of E-cadheren was significantly increased (P<0.01). However, compared with the dexamethasone injection group, in the compound Kushen injection group, the pathological changes of lung tissues were similar, and the expression levels of E-cadheren, Vimentin, TGF-β1, Smad3, GSK-3β and β-cateninin were not significantly different. Conclusion::Compound Kushen injection can alleviate pulmonary fibrosis of lung in the treatment of RIPI, and the mechanism may be associated with inhibiting the mRNA and protein expressions of TGF-β1, Smad3, GSK-3β and β-catenin related to epithelial-mesenchymal transition(EMT), promoting the expression of E-cadheren, and inhibiting the expression of Vimentin, so as to inhibit the occurrence of EMT.

Effect of Tianwang Buxin Pills on Behavior, HPA Axis, BDNF Expression and Phosphorylation of GSK3β in Mice with Chronic Unpredictable Stress / 中国实验方剂学杂志

Objective:To investigate the effect of Tianwang Buxin pills on behavior， hypothalamus pituitary adrenal axis （HPA axis）， hippocampal glycogen synthase kinase 3β （GSK3β） phosphorylation and brain-derived neurotrophic factor （BDNF） expression in mice with chronic unpredictable stress， and explore its mechanisms for antidepressant-like action. Method:Totally 60 male ICR mice were randomly divided into normal group， chronic stress model group， fluoxetine hydrochloride group （10 mg·kg-1） and Tianwang Buxin pills high， middle and low dose groups （3.6， 1.8， 0.9 g·kg-1）. The mice were subjected to the chronic unpredictable stress （CUS） protocol for a period of 28 d to induce depressive-like behavior. Then， a sucrose preference test， open-field test and novelty-suppressed feeding test were performed to detect the behavior changes. The blood， adrenal gland and hippocampus of mice were collected. The contents of adrenocorticotropic hormone （ACTH） and corticosterone （CORT） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. The changes of GSK3β phosphorylation and BDNF expression in hippocampus were detected by Western blot， and the adrenal index was then calculated. Result:As compared with the normal group， the sucrose water preference was significantly decreased （P<0.01）， the number of opening activities was significantly reduced （P<0.05）， the feeding latency of novelty inhibition was prolonged （P<0.01）， the serum ACTH and CORT contents were significantly increased （P<0.05，P<0.01）， GSK3β phosphorylation and BDNF expression levels in hippocampus were significantly decreased （P<0.01）， and adrenal index was significantly increased in model group （P<0.01）. As compared with the model group， Tianwang Buxin pills treatment significantly reversed the CUS-induced behavioral abnormalities in depression model mice （P<0.05， P<0.01）， significantly decreased the levels of plasma ACTH and CORT （P<0.01） and adrenal and adrenal gland index （P<0.01）， while increased GSK3β phosphorylation and BDNF expression in hippocampus （P<0.05， P<0.01）， with its effect similar to that of fluoxetine hydrochloride. Conclusion:Tianwang Buxin pills produced antidepressant-like effects in chronic unpredictable stress mice， and its mechanism may be associated with inhibiting HPA axis activity and up-regulating GSK3β phosphorylation and BDNF protein expression in hippocampus.

Effect of Chaihu Jia Longgu Mulitang on Signaling Pathway of PI3K/Akt/GSK3β/β-catenin in Hippocampus Tissue of Rats with Depression / 中国实验方剂学杂志

Objective:To study the effect of Chaihu Jia Longgu Mulitang on phosphatidylinositol-3-kinases （PI3K）/protein kinase B （Akt）/glycogen synthase kinase 3β （GSK3β）/β-catenin signaling pathway of hippocampus in rats with depression. Method:A total of 120 SD rats were randomly divided into normal group，model group， and low， middle and high-dose Chaihu Jia Longgu Mulitang groups（3.25，6.5，13 g·kg-1）， and fluoxetine group， with 20 rats in each group. Except normal group， the depression model was prepared through chronic unpredictable mild stimulation（CUMS）. The normal group and the model group were given normal saline with 6.5 g·kg-1 by gavage. Chaihu Jia Longgu Mulitang groups were intragastrically given corresponding herbal drugs 3.75，6.5，13 g·kg-1， while fluoxetine group was intragastrically given fluoxetine 10 mg·kg-1 for 21 days， once a day. Then the depressive behaviors of rats were observed by sucrose preference test （SPT） and forced swimming test （FST）. Western blot was used to detect the protein expressions of PI3K，Akt，GSK3β and phosphorylation level. Result:Compared with normal group，the sucrose preference index was decreased significantly，while the immobility time in FST was increased significantly（P<0.01）， the protein expressions of PI3K， p-PI3K p110， p-PI3K p85 were decreased significantly （P<0.01）， and expressions of Akt， p-Akt Thr308，p-Akt Ser473， p-GSK3β Ser9 and β-catenin were decreased significantly（P<0.01）， while the level of GSK3β， p-GSK3β Tyr216 were increased significantly in model group（P<0.05，P<0.01）. Compared with model group，Chaihu Jia Longgu Mulitang could increase sucrose preference index and decrease the immobility time in FST（P<0.01）， the protein expressions of PI3K p110 and PI3K p85 was increased significantly （P<0.01）， levels of Akt Thr308，Akt Ser473， p-GSK3β Ser9， β-catenin were increased significantly （P<0.01）， whereas levels of GSK3β， and GSK3β Tyr216 were decreased significantly. Conclusion:Chaihu Jia Longgu Mulitang could increase protein expression and activity of PI3K in rat hippocampus， activate Akt， inhibit GSK3β kinase activity and prevent β-catenin from degradation， so as to increase PI3K/Akt pathway activity in rat hippocampus， and protect hippocampal neurons.

Effect of Hei Xiaoyaosan on Expressions of Aβ1-42, GSK-3β, NEP, IDE in Hippocampus Area of Alzheimer's Dementia Mice / 中国实验方剂学杂志

Objective: To observe the effect of Hei Xiaoyaosan on expressions of β-amyloid 1-42 peptide(Aβ1-42),glycogen synthase kinase-3β(GSK-3β),neprilysin(NEP),insulin-degrading enzyme(IDE) in the hippocampus area of Alzheimer's dementia mice. Method: After weighing, 42 APP/PSI bivalent transgenic mice were randomly divided into 4 groups:10 mice in the model group, 10 mice in the positive drug control group, 11 mice in the high-dose Hei Xiaoyaosan group, and 11 mice in the low-dose Hei Xiaoyaosan group; 10 wild C57BL/6J mice of the same age and strain were used for negative control group. Drugs were administered to mice by gavage once a day for 12 weeks. Then the behavior of all the mice were detected by Morris water maze, the morphological changes in hippocampal neurons were observed by hematoxylineosin(HE) staining, the expressions of Aβ1-42, GSK-3β, NEP and IDE proteins in hippocampus were detected by immunohistochemistry. Result: After 3 months of treatment, compared with negative control groups, the average escaping latency periods prolonged significantly, and the number of cross-platform was decreased significantly in model group (Pβ1-42 and GSK-3β proteins in model mice hippocampus were significantly increased (PPPβ1-42 and GSK-3β proteins in the hippocampus of drug groups were significantly decreased (PPPConclusion: Hei Xiaoyaosan can significantly improve the learning and memory abilities of AD mice, which may be related to the reduction of cognitive impairment in AD mice by regulating abnormal deposition and degradating Aβ in the hippocampus.

Effect of Puerarin in Reducing Insulin Resistance in HepG2 Cells via PI3K/Akt/GSK-3β Signal Transduction Pathway / 中国实验方剂学杂志

Objective: To observe the effect of puerarin on phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β) in insulin resistant HepG2 cells. Method: HepG2 cells were treated with palmitic acid 0.5 mmol·L-1 and insulin 9×10-4 U·L-1 to induce insulin resistant condition for 24 h. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay to determine the concentration of puerarin. This experiment included normal control group, model control group and puerarin groups of different doses (40, 80, 160,320 μg·L-1). Glucose detection kit was used to detect the content of glucose in cell culture supernatant. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels in supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay (ELISA). Hepatic glycogen assay kit was used for detecting the hepatic glycogen content in HepG2 cells. Western blot was applied to detect protein expression levels of PI3K, Akt, p-Akt, GSK-3β and p-GSK-3β. Result: Compared with those in the normal control group, the glucose consumption rate was significantly down-regulated in HepG2 cells in the model control group (PPα and IL-6 were increased in supernatant of cell culture medium (PPβ protein expression was up-regulated (PPα and IL-6 were reduced in supernatant of cell culture medium (PPβ protein expression was down-regulated, but its phosphorylation inactivation was increased (PConclusion: Puerarin alleviates the insulin resistance of HepG2 cells by strengthening the PI3K/Akt/GSK-3β signal transduction process and increasing the glycogen content in hepatocytes.

Effect and Mechanism of Jiaotaiwan on Cognitive Impairment in Diabetic Mice / 中国实验方剂学杂志

Objective: To explore effect and mechanism of Jiaotaiwan (JTW) on cognitive impairment in diabetic mice. Method: The 40 db/db mice of spontaneous diabetes were randomly divided into model group, JTW 1 group (1.68 g·kg-1), JTW 2 group (3.36 g·kg-1), and JTW 3 group (8.40 g·kg-1), with 8 mice in each group, and 8 C57BL/6J mice were included into normal group. After 8 weeks of treatment, behavioral test,fasting blood glucose (FBG), fasting insulin (Fins), insulin resistance index (HOMA-IR), total triglyceride (TG), total cholesterol (TC), free fatty acids (FFA), high density lipoprotein (HDL), and low density lipoprotein (LDL) were detected. Western blot was used to detect protein expressions of pSer199, pSer202, pSer214, p-Akt, glycogen synthase kinase-3β(GSK-3β), phospho-GSK-3β(p-GSK-3β). Result: Compared with model group, FBG, Fins, HOMA-IR, TG, TC, HDL, FFA in JTW group decreased significantly (P0.01), with a significant improvement in cognitive function (P0.01), protein expressions of taupSer199, pSer202, pSer214 and GSK-3β in hippocampus of mice decreased significantly (PP0.01), and expressions of p-Akt and p-GSK3 beta significantly increased (P0.01). Conclusion: JTW 3 (Coptidis Rhizoma-Cinnamomi Cortex 10:1) can alleviate insulin resistance and cognitive impairment. The mechanism may be related to the inhibition of over-phosphorylation of tau protein, the down-regulation of GSK-3β protein expression and the up-regulation of p-Akt and p-GSK-3β expressions.

Effect of Wendantang on PI3K,Akt and GSK3β of Hippocampus in Rats with Schizophrenia / 中国实验方剂学杂志

Objective: To research the effect of Wendantang on phosphatidylinositol-3-kinases(PI3K)/protein kinase B(Akt)/glycogen synthase kinase 3β(GSK3β) signaling pathway of hippocampus in rats with schizophrenia. Method: Sixty SD rats were randomly divided six groups:normal group (A), model group (B), Clozapine group (C), high-dosage group Wendantang (D), medium-dosage Wendantang group (E) and low-dosage Wendantang group (F), with 10 rats in each group. The rats of normal group and model group were given normal saline. The rats of Clozapine group were given 20 mg·kg-1 Clozapine. The rats of high-dosage, medium-dosage, low-dosage Wendantang groups were respectively given 40,20,10 g·kg-1 Wendantang, once a day, for 21 days.Two hours later after the last administration with Wendantang or saline,except for group A, groups B,C,D,E,F were intraperitoneally given MK-801 0.6 mg·kg-1 to establish the rat schizophrenia model. After modeling, the changes in stereotyped behavior of each group were observed and recorded. After three days,the rats were put to death,and the hippocampus tissue were tested. According to Sams Dodd and Hoffman's standard, the stereotyped behavior of rats was scored. The protein expressions of PI3K, Akt and GSK3β of hippocampus were determined by Western blot. The mRNA expressions of PI3K, Akt and GSK3β of hippocampus were tested by Real-time PCR. Result: Compared with A, the stereotyped behavior score of the B was significantly increased (Pβ protein and mRNA of hippocampus were significantly decreased (PPβ protein and PI3K, Akt and GSK3β mRNA of hippocampus (PPConclusion: Wendantang could regulate the PI3K/Akt/GSK3 signaling pathway by improving the stereotyped behavior and increasing the expressions of PI3K, Akt, GSK3β of hippocampus in rats with schizophrenia, so as to achieve the purpose of treating schizophrenia.

ABT-263 induced A431 cell apoptosis and its effect on AKT downstream signaling pathway / 中国职业医学

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.

Effect of water extract from Jiangtang Decoction on PI3K/Akt signal pathway of skeletal muscle metabolism in KK-Ay diabetic mice / 药物评价研究

Objective To explore the effects of water extract from Jiangtang Decoction (WEJTD) on PI3K/Akt signal pathway of skeletal muscle metabolism in KK-Ay diabetic mice.Methods Totally 50 KK-Ay mice were randomly divided into five groups:model group,metformin (positive drug,250 mg/kg) group,WEJTD low,medium,and high dose (2,4,and 8 g/kg) group,with 10 C57BL/6J mice as normal group.The relative drugs were ig administered once a day for 12 weeks,and mice in control group and model group were perfused with distilled water of equal volume.After 12 weeks' oral administration,mice were executed to separate serum,serum insulin level was detected by ELISA kit method;RNA was extracted from muscle tissue by Trizol,and real-time PCR were used to detect the level of PI3K,Akt,GLUT-4,GSK-3β,GS and IRS-1 mRNA.Results WEJTD can down-regulate concentration of insulin in serum and GSK-3β mRNA in skeletal muscle (P ＜ 0.05 and 0.001),and down-regulate mRNA of PI3K,Akt,IRS-1,GLUT-4,and GS in skeletal muscle (P ＜ 0.05,0.01,and 0.001).Conclusion WEJTD decreased glycogen deposition and stimulated glucose transport in skeletal muscle through upregulation of PI3K/Akt signaling pathway.

Effects and mechanisms of different frequencies of electroacupuncture for learning and memory ability of Alzheimer's rats / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of different frequencies of electroacupuncture (EA) at "Baihui (GV 20)" and "Shenshu(BL 23)" for the learning and memory ability as well as glycogen synthase kinase-3β (GSK-3β) and growth associated protein-43 (GAP-43) in hippocampal tissue of rats with Alzheimer's disease(AD), so as to explore the mechanism of different frequencies of EA for the prevention and treatment of AD.</p><p><b>METHODS</b>One hundred and twelve healthy Wistar male rats were divided into seven groups by random number table, namely a normal group, a sham operation group, a model group, an acupuncture group, a 2 Hz EA group, a 30 Hz EA group, and a 50 Hz EA group, 16 rats in each one. The rats in the normal group were conventionally raised in the laboratory without any treatment. 0.9% NaCl solution was injected into bilateral dentate convolution of hippocampus in rats of the sham operation group. AD model was established by β-amyloid protein1-42 (Aβ1-42) injected into bilateral dentate convolution of hippocampus in the other groups. 15 days after establishment, no treatment was applied in the model and sham operation groups, and EA with corresponding frequencies at "Baihui (GV 20)" and "Shenshu (BL 23)" was used in the three EA groups for 2 sessions, once a day and 7 times as one session. There was 1 day between the two sessions. The same acupoints were adopted in the acupuncture group, without electrical connection. The escape latency, the first spanning platform time, and the number of crossing platform were tested in the Morris water maze immediately after treatment. The expressions of GSK-3β and GAP-43 were examined by immunohistochemistry and Western blot.</p><p><b>RESULTS</b>①Morris water maze tests showed that the escape latency and the first spanning platform time significantly increased in the model group compared with those in the normal group (both<0.01), and the number of crossing platform decreased (<0.01). Compared with the model group, the escape latency and the first spanning platform times decreased in the acupuncture and three EA groups (all<0.01), and the numbers of crossing platform increased (<0.01). Compared with the acupuncture and 2 Hz, 30 Hz EA groups, the escape latency decreased in the 50 Hz EA group (<0.01,<0.05); the first spanning platform time reduced (all<0.01); the number of crossing platform increased (<0.01,<0.05). ②The expressions of GSK-3β and GAP-43 of the model group increased compared with those of the normal group(both<0.01). The expressions of GSK-3β in the acupuncture and three EA groups decreased compared with that in the model group (all<0.01), and the expressions of GAP-43 increased (all<0.01). The expressions of GSK-3β decreased and GAP-43 increases in the 50 Hz EA group compared with those in the acupuncture group and 2 Hz, 30 Hz groups (all<0.01).</p><p><b>CONCLUSIONS</b>EA may promote synaptic damage rehabilitation by down regulating GSK-3β and up regulating GAP-43 to improve learning and memory ability of AD rats. The effect of 50 Hz EA is better than those of 30 Hz and 2 Hz EA and acupuncture.</p>