Impact of salvianolic acid B on cognitive function and GSK-3β/β-Catenin signaling pathway in rats with post-traumatic stress disorder / 天津医药

Objective To investigate whether salvianolic acid B(Sal B)can improve the cognitive function in rats with post-traumatic stress disorder(PTSD)by regulating GSK-3β/β-Catenin signal pathway.Methods Sixty rats were randomly grouped into the normal group,the PTSD group,the Sal B low-dose group(10 mg/kg),the Sal B high-dose group(20 mg/kg)and the GSK-3β inhibitor group(30 mg/kg CHIR-99021),with 12 rats in each group.In addition to the normal group,rats in other groups were constructed PTSD rat models by using single prolonged stress(SPS)method.Open field test and Morris water maze test were applied to evaluate the cognitive function of rats.Nissl staining was applied to observe the pathological changes of hippocampal neurons.TUNEL staining was applied to detect the apoptosis of hippocampal neurons.Western blot assay was applied to detect the expression of cleared caspase-3,B-cell lymphoma gene-2-associated X protein(Bax),proto-oncogene(c-Myc),Cyclin D1,total GSK-3β(t-GSK-3β),phosphorylated GSK-3β(p-GSK-3β),total β-Catenin(t-β-Catenin)and phosphorylated β-catenin(p-β-Catenin)proteins in hippocampus.Results Compared with the PTSD group,the number of crawling spaces,standing times,total movement distance and times of crossing the original platform of rats were higher in the Sal B low-dose group,the Sal B high-dose group and the GSK-3β inhibitor group.The escape latency and the time to cross the original platform for the first time were shorter,the apoptosis rate of hippocampal neurons and the expression levels of Bax,cleaved caspase-3,t-GSK-3β and p-β-Catenin proteins in hippocampus were lower,and the expression levels of Cyclin D1,c-Myc,p-GSK-3β,t-β-Catenin proteins were higher(P＜0.05).Conclusion Sal B can reduce the apoptosis and damage of hippocampal neurons in rats with PTSD and improve cognitive dysfunction in rats,and inhibit the GSK-3β/β-Catenin signal pathway.

Application Study of Enzyme Inhibitors and Their Conformational Optimization in The Treatment of Alzheimer’s Disease / 生物化学与生物物理进展

Alzheimer’s disease (AD) is a central neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment, and there is a lack of effective drugs to treat AD clinically. Existing medications for the treatment of AD, such as Tacrine, Donepezil, Rivastigmine, and Aducanumab, only serve to delay symptoms and but not cure disease. To add insult to injury, these medications are associated with very serious adverse effects. Therefore, it is urgent to explore effective therapeutic drugs for AD. Recently, studies have shown that a variety of enzyme inhibitors, such as cholinesterase inhibitors, monoamine oxidase (MAO)inhibitors, secretase inhibitors, can ameliorate cholinergic system dysfunction, Aβ production and deposition, Tau protein hyperphosphorylation, oxidative stress damage, and the decline of synaptic plasticity, thereby improving AD symptoms and cognitive function. Some plant extracts from natural sources, such as Umbelliferone, Aaptamine, Medha Plus, have the ability to inhibit cholinesterase activity and act to improve learning and cognition. Isochromanone derivatives incorporating the donepezil pharmacophore bind to the catalytic active site (CAS) and peripheral anionic site (PAS) sites of acetylcholinesterase (AChE), which can inhibit AChE activity and ameliorate cholinergic system disorders. A compound called Rosmarinic acid which is found in the Lamiaceae can inhibit monoamine oxidase, increase monoamine levels in the brain, and reduce Aβ deposition. Compounds obtained by hybridization of coumarin derivatives and hydroxypyridinones can inhibit MAO-B activity and attenuate oxidative stress damage. Quinoline derivatives which inhibit the activation of AChE and MAO-B can reduce Aβ burden and promote learning and memory of mice. The compound derived from the combination of propargyl and tacrine retains the inhibitory capacity of tacrine towards cholinesterase, and also inhibits the activity of MAO by binding to the FAD cofactor of monoamine oxidase. A series of hybrids, obtained by an amide linker of chromone in combine with the benzylpiperidine moieties of donepezil, have a favorable safety profile of both cholinesterase and monoamine oxidase inhibitory activity. Single domain antibodies (such as AAV-VHH) targeted the inhibition of BACE1 can reduce Aβ production and deposition as well as the levels of inflammatory cells, which ultimately improve synaptic plasticity. 3-O-trans-p-coumaroyl maslinic acid from the extract of Ligustrum lucidum can specifically inhibit the activity of γ-secretase, thereby rescuing the long-term potentiation and enhancing synaptic plasticity in APP/PS1 mice. Inhibiting γ-secretase activity which leads to the decline of inflammatory factors (such as IFN-γ, IL-8) not only directly improves the pathology of AD, but also reduces Aβ production. Melatonin reduces the transcriptional expression of GSK-3β mRNA, thereby decreasing the levels of GSK-3β and reducing the phosphorylation induced by GSK-3β. Hydrogen sulfide can inhibitGSK-3β activity via sulfhydration of the Cys218 site of GSK-3β, resulting in the suppression of Tau protein hyperphosphorylation, which ameliorate the motor deficits and cognitive impairment in mice with AD. This article reviews enzyme inhibitors and conformational optimization of enzyme inhibitors targeting the regulation of cholinesterase, monoamine oxidase, secretase, and GSK-3β. We are hoping to provide a comprehensive overview of drug development in the enzyme inhibitors, which may be useful in treating AD.

Down-regulating GSK3β alleviates hypoxia/reoxygenation-induced injury of senescent renal tubular epithelial cells by inhibiting the function of ITPR1-GRP75-VDAC1 complex / 器官移植

<b>Objective</b> To evaluate the effect of glycogen synthase kinase 3β (GSK3β) on hypoxia/reoxygenation (H/R)-induced injury of senescent renal tubular epithelial cell (RTEC) in aged mice and its regulatory mechanism. <b>Methods</b> RTEC were divided into the Young group (young RTEC with normal growth), Old group (aged RTEC induced by Etoposide), Old+Ad-shNC+H/R group [aged RTEC induced by Etoposide and then transfected with adenovirus negative control (Ad-shNC) for H/R treatment], and Old+Ad-shGSK3β+H/R group (aged RTEC induced by Etoposide and then transfected with short-hairpin RNA-expressing adenovirus with targeted silencing GSK3β for H/R treatment), respectively. Apoptosis level and mitochondrial reactive oxygen species level were detected by flow cytometry. Calcium ion level was determined by immunofluorescence staining. The expression and phosphorylation levels of GSK3β, mitochondria-associated endoplasmic reticulum membrane (MAM)-related proteins of inositol 1,4,5-trisphosphate receptor1 (ITPR1), voltage dependent anion-selective channel 1(VDAC1) and glucose-regulated protein 75 (GRP75) were detected by Western blot. The interaction between GSK3β and MAM-related proteins was analyzed by immunoprecipitation. <b>Results</b> Compared with the Young group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old group. Compared with the Old group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were lower in the Old+Ad-shGSK3β+H/R group, and the differences were statistically significant (all <i>P</i><0.05). Compared with the Young group, the expression levels of ITPR1, GRP75 and GSK3β proteins were up-regulated, the phosphorylation levels of ITPR1 and GRP75 were increased, whereas the total protein and phosphorylation levels of VDAC1 were decreased in the Old group. Compared with the Old group, the expression level of GSK3β protein was unchanged, the total protein and phosphorylation levels of ITPR1 and GRP75 were increased, the expression level of total VDAC1 protein remained unchanged and the phosphorylation level was increased in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the expression level of GSK3β protein was decreased, the expression levels of total ITPR1, GRP75 and VDAC1 proteins were unchanged, whereas the phosphorylation levels were decreased in the Old+Ad-shGSK3β+H/R group. Immunoprecipitation showed that GSK3β could interact with ITPR1, GRP75 and VDAC1 proteins. <b>Conclusions</b> The expression level of GSK3β is up-regulated in senescent RTEC. Down-regulating GSK3β expression may reduce the phosphorylation level of ITPR1-GRP75-VDAC1 complex, constrain the overload of mitochondrial calcium ion, protect mitochondrial function and mitigate cell damage during reperfusion.

Inhibitory effect of royal jelly acid on proliferation of human colon cancer SW620 cells and its network pharmacological analysis / 吉林大学学报(医学版)

Objective:To discuss the effect of royal jelly acid(10-HDA)on the proliferation and migration of the human colon cancer SW620 cells based on the network pharmacology,and to clarify its related molecular mechanism.Methods:The active ingredients such as 10-HDA and their corresponding targets were retrieved by using the keyword"royal jelly"from the Traditiomal Chinese Medicine Systems Pharmacology(TMSCP)Database and the Traditiomal Chinese Medicine Integrated Database(TCMID);the small molecule targets were predicted by the Swiss Target Prediction Database.The GeneCards Database and the Online Mendelian Inheritance in Man(OMIM)Database were used to obtain the targets with the keyword"Colon Cancer";the protein-protein interaction(PPI)network was constructed by using the String Database and Cytoscape 3.8.0 Software to screen the core targets;the Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis were analyzed by Metascape Database;the specific ingredient 10-HDA was screened for the in vitro activity experiments.The human colon cancer SW620 cells with good growth status were divided into control group and different doses(1,5,10,15,and 20 mmol·L-1)of 10-HDA groups.The viabilities of the cells in various groups were detected by MTT method and the survival rates of the cells were calculated.The SW620 cells were divided into control group,low dose(5 mmol·L-1)of 10-HDA group,middle dose(10 mmol·L-1)of 10-HDA group,and high dose(15 mmol·L-1)of 10-HDA group;Hoechst33342 staining method was used to observe the morphology of the cells in various groups;cell scratch test was used to detect the scratch healing rates of the cells in various groups;flow cytometry was used to detect the percentages of the cells at different cell cycles in various groups;biochemical method was used to detect the activities of total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)in the cells in various groups;Western blotting method was used to detect the expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine-containing aspartate proteolytic enzyme-3(Caspase-3),cysteine-containing aspartate proteolytic enzyme-9(Caspase-9),glycogen synthase kinase 3β(GSK3β),β-catenin,and cyclin D1 proteins in the cells in various groups.Results:Six active ingredients of royal jelly were screened out by the TCMSP Database,and 28 core targets of 10-HDA in the treatment of colon cancer were obtained.The GO function enrichment analysis mainly included the signaling pathways such as cell proliferation and apoptosis.The KEGG signaling pathway enrichment analysis included the cell cycle,prostate cancer,cell senescence,and p53 signaling pathways;the GSK3β/β-catenin signaling pathway was closely related to the cell cycle.Compared with control group,the viabilities of the cells in 5,10,15,and 20 mmol·L-110-HDA groups were decreased in a dose-dependent manner(P<0.05 or P<0.01),the numbers of apoptotic cells in different doses of 10-HDA groups were significantly increased,and the scratch healing rates of the cells were significantly decreased(P<0.05 or P<0.01);the percentages of the cells at S phase in middle and high doses of 10-HDA groups were significantly increased(P<0.05 or P<0.01),the activities of T-AOC and SOD in the cells in different doses of 10-HDA groups were significantly decreased(P<0.05 or P<0.01).Compared with control group,the expression level of Bcl-2 protein in the cells in low dose of 10-HDA group was significantly decreased(P<0.01),and the expression level of GSK3β protein was significantly increased(P<0.05);compared with control group,the expression levels of Bax,Caspase-3,Caspase-9,and GSK3β proteins in the cells in middle and high doses of 10-HDA groups were significantly increased(P<0.05 or P<0.01),and the expression levels of Bcl-2,β-catenin,and CyclinD1 proteins were significantly decreased(P<0.01).Conclusion:10-HDA can significantly inhibit the proliferation and migration of the colon cancer cells and promote the apoptosis and oxidation levels of the colon cancer cells,and its mechanism may be related to the activation of the GSK3β/β-catenin signaling pathway.

Mechanism of ellagic acid improving cognitive dysfunction in APP/PS double transgenic mice based on PI3K/AKT/GSK-3β signaling pathway / 中国药理学通报

Aim To investigate the effect of ellagic acid (EA) on cognitive function in APP/PS 1 double- transgenic mice, and to explore the regulatory mechanism of ellagic acid on the level of oxidative stress in the hippocampus of double-transgenic mice based on the phosphatidylinositol 3-kinase/protein kinase B/glycogen synthase kinase-3 (PI3K/AKT/GSK-3 β) signaling pathway. Methods Thirty-two SPF-grade 6-month-old APP/PS 1 double transgenic mice were randomly divided into four groups, namely, APP/PS 1 group, APP/PS1 + EA group, APP/PS1 + LY294002 group, APP/PS 1 + EA + LY294002 group, with eight mice in each group, and eight SPF-grade C57BL/6J wild type mice ( Wild type) were selected as the blank control group. The APP/PS 1 + EA group was given 50 mg · kg

Effect of Qiwei Baizhusan on Cognitive Dysfunction in Rats with Diabetic Encephalopathy Based on PI3K/Akt/GSK-3β Signaling Pathway / 中国实验方剂学杂志

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.

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.

Glycogen synthase kinase 3 inhibition enhances mineral nodule formation by cementoblasts in vitro

Abstract This study aimed to investigate whether GSK-3 inhibition (CHIR99021) effectively promoted mineralization by cementoblasts (OCCM-30). OCCM-30 cells were used and treated with different concentrations of CHIR99021 (2.5, 5, and 10 mM). Experiments included proliferation and viability, cellular metabolic activity, gene expression, and mineral nodule formation by Xylene Orange at the experimental time points. In general, CHIR99021 did not significantly affect OCCM-30 viability and cell metabolism (MTT assay) (p > 0.05), but increased OCCM-30 proliferation at 2.5 mM on days 2 and 4 (p < 0.05). Data analysis further showed that inhibition of GSK-3 resulted in increased transcript levels of Axin2 in OCCM-30 cells starting as early as 4 h, and regulated the expression of key bone markers including alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx-2), osteocalcin (Ocn), and osterix (Osx). In addition, CHIR99021 led to an enhanced mineral nodule formation in vitro under both osteogenic and non-osteogenic conditions as early as 5 days after treatment. Altogether, the results of the current study suggest that inhibition of GSK-3 has the potential to promote cementoblast differentiation leading to increased mineral deposition in vitro.

Role of TIPE2 in sepsis-induced myocardial injury in mice: the relationship with AKT/GSK-3β/β-catenin signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of tumor necrosis factor-alpha-induced protein-8 like-2(TIPE2) in sepsis-induced myocardial injury and the relationship with serine-threonine kinase(AKT)/glycogen synthase kinase-3β(GSK-3β)/β-catenin signaling pathway in mice.Methods:Sixteen male wild-type C57BL/6N mice and 16 TIPE2-gene knockout C57BL/6N mice, aged 6-8 weeks, with a body mass index of 20-25 g, were divided into 4 groups using a random number table method: wild-type+ sham operation group(group WT-sham), wild-type+ cecal ligation and perforation(CLP) group(group WT-CLP), TIPE2-gene knockout sham operation group(group KO-sham) and TIPE2-gene knockout CLP group(group KO-CLP), with 8 mice in each group. The model of myocardial injury induced by sepsis was developed by CLP in anesthetized animals. Blood samples from the inferior vena cava were collected at 24 h after surgery for determination of the concentrations of cardiac troponin I(cTnI) in serum by enzyme-linked immunosorbent assay. Then the mice were sacrificed and myocardial tissues were collected for determination of the pathological changes(by hematoxylin and eosin staining), expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6 mRNA(by quantitative polymerase chain reaction), and expression of TIPE2, phosphorylated AKT(p-AKT), phosphorylated GSK-3β(p-GSK-3β) and β-catenin(by Western blot).Results:Compared with the corresponding Sham groups, the serum cTnI concentration was significantly increased, the expression of TNF-α, IL-1β and IL-6 mRNA and expression of p-AKT, p-GSK-3β and β-catenin in myocardial tissues were up-regulated, the expression of TIPE2 was down-regulated( P<0.05), and the pathological changes of myocardium were found in corresponding CLP groups. Compared with group WT-CLP, the serum cTnI concentration was significantly increased, the expression of TNF-α, IL-1β and IL-6 mRNA and expression of p-AKT, p-GSK-3β and β-catenin in myocardial tissues were up-regulated, the expression of TIPE2 was down-regulated( P<0.05), and the pathological changes of myocardium were aggravated in group KO-CLP( P<0.05). Conclusions:TIPE2 reduces the myocardial injury probably through inhibiting the AKT/GSK-3β/β-catenin signaling pathway in septic mice.

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.

Genome-wide identification of GSK gene family in Senna tora L. and expression analyses / 药学学报

Glycogen synthase kinase 3/SHAGGY-like kinase (GSK3) proteins play important roles in regulating plant growth, development, and stress response. In order to reveal the characteristics of GSK family members in the medicinal plant Senna tora L., in this study, we conducted the identification and expression analyses of GSKs in S. tora based on its whole genome data, combined with bioinformatics and gene expression research methods. The results showed that a total of nine S. tora GSK genes were identified, all of which contained the GSK characteristic kinase domains. All members were distributed on six chromosomes, the encoding amino acid length ranged from 465 to 943 aa, the protein molecular weight was from 33.57 to 88.83 kDa, and the average isoelectric point was 8.2. The StoSKs were divided into four evolutionary branches, and the StoSKs in the same evolutionary branch shared the same exon/intron structure and conserved motifs. The expansion of the StoSKs gene family was mainly due to segment duplication events, and there were 17, 11, 8 and 7 pairs of collinear genes with Glycine max, Medicago truncatula, Arabidopsis thaliana and Oryza sativa, respectively. The promoter regions of StoSKs mostly contained responses elements related to stress stimulation, growth and development, and hormone induction. Transcriptome data analysis showed that StoSKs were expressed in different tissues, with the highest expression level in roots. Quantitative real-time PCR (qRT-PCR) analysis indicated that StoSKs in different evolutionary branches displayed a synergistic expression pattern response to light, and most of StoSKs could rapidly respond to NaCl stress with significantly up-regulated expression. All the results provide a basis for further analysis of the biological functions of the GSKs gene family in S. tora.

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.

Effect of aluminum exposure on abnormal phosphorylationof tau protein in PC12 cells of rats / 预防医学

Objective @#To investigate the effect of aluminum exposure on expression of miR-497-5p, wingless murine breast cancer virus integration site family member 3a (Wnt3a), β-catenin protein, glycogen synthase kinase-3β (GSK-3β) protein and tau protein in rat adrenal pheochromocytoma PC12 cells, so as to provide insight into unraveling the mechanisms underlying aluminum exposure-induced abnormal phosphorylation of tau protein.@* Methods@# PC12 cells were exposed to Al(mal)3 at concentrations of 0, 100, 200, 400 μmol/L for 24 h. The viability of PC12 cells was measured using cell counting kit-8 (CCK-8) assay. The relative expression of miR-497-5p and Wnt3a was detected using a real-time fluorescent quantitative PCR (RT-qPCR) assay, and the expression of Wnt3a, β-catenin, GSK-3β, P-GSK-3β (Ser9), tau and p-tau (Ser396) proteins were determined using Western blotting. @*Results @#The viability of PC12 cells appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=323.473, P=0.001). RT-qPCR assay detected that the relative miR-497-5p expression appeared a tendency towards a rise with the increase of aluminum dose (Ftrend=14.888, P=0.031), and the relative Wnt3a expression appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=165.934, P<0.001). The miR-497-5p expression negatively correlated with the relative Wnt3a expression (r=-0.693, P=0.012). The expression of Wnt3a (Ftrend=357.656, P=0.001), β-catenin (Ftrend=208.750, P=0.001) and p-GSK-3β (Ser9) proteins (Ftrend=512.583, P<0.001) appeared a tendency towards a decline with the increase of aluminum dose, and the expression of GSK-3β (Ftrend=39.965, P<0.001), tau (Ftrend=277.929, P=0.006) and p-tau (Ser396) proteins (Ftrend=96.247, P=0.002) appeared a tendency towards a rise with the increase of aluminum dose. @*Conclusion@# Up-regulation of miR-497-5p and GSK-3β expression and down-regulation of Wnt3a and β-catenin expression may be a mechanism underlying aluminum exposure-induced abnormal phosphorylation of tau protein.

Baicalin Ameliorates Corticosterone-Induced Depression by Promoting Neurodevelopment of Hippocampal via mTOR/GSK3β Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the role of hippocampal neurodevelopment in the antidepressant effect of baicalin.@*METHODS@#Forty male Institute of Cancer Research mice were divided into control, corticosterone (CORT, 40 mg/kg), CORT+baicalin-L (25 mg/kg), CORT+baicalin-H (50 mg/kg), and CORT+fluoxetine (10 mg/kg) groups according to a random number table. An animal model of depression was established by chronic CORT exposure. Behavioral tests were used to assess the reliability of depression model and the antidepressant effect of baicalin. In addition, Nissl staining and immunofluorescence were used to evaluate the effect of baicalin on hippocampal neurodevelopment in mice. The protein and mRNA expression levels of neurodevelopment-related factors were detected by Western blot analysis and real-time polymerase chain reaction, respectively.@*RESULTS@#Baicalin significantly ameliorated the depressive-like behavior of mice resulting from CORT exposure and promoted the development of dentate gyrus in hippocampus, thereby reversing the depressive-like pathological changes in hippocampal neurons caused by CORT neurotoxicity. Moreover, baicalin significantly decreased the protein and mRNA expression levels of glycogen synthase kinase 3β (GSK3β), and upregulated the expression levels of cell cycle protein D1, p-mammalian target of rapamycin (mTOR), doublecortin, and brain-derived neurotrophic factor (all P<0.01). There were no significant differences between baicalin and fluoxetine groups (P>0.05).@*CONCLUSION@#Baicalin can promote the development of hippocampal neurons via mTOR/GSK3β signaling pathway, thus protect mice against CORT-induced neurotoxicity and play an antidepressant role.

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.

Glycogen synthase kinase-3 (GSK-3) a magic enzyme: it's role in diabetes mellitus and glucose homeostasis, interactions with fluroquionlones. A mini-review / Glicogênio sintase quinase-3 (GSK-3), uma enzima mágica: seu papel no diabetes mellitus e na homeostase da glicose: interações com fluoroquinolonas. Uma mini-revisão

Abstract Diabetes mellitus (DM) is a non-communicable disease throughout the world in which there is persistently high blood glucose level from the normal range. The diabetes and insulin resistance are mainly responsible for the morbidities and mortalities of humans in the world. This disease is mainly regulated by various enzymes and hormones among which Glycogen synthase kinase-3 (GSK-3) is a principle enzyme and insulin is the key hormone regulating it. The GSK-3, that is the key enzyme is normally showing its actions by various mechanisms that include its phosphorylation, formation of protein complexes, and other cellular distribution and thus it control and directly affects cellular morphology, its growth, mobility and apoptosis of the cell. Disturbances in the action of GSK-3 enzyme may leads to various disease conditions that include insulin resistance leading to diabetes, neurological disease like Alzheimer's disease and cancer. Fluoroquinolones are the most common class of drugs that shows dysglycemic effects via interacting with GSK-3 enzyme. Therefore, it is the need of the day to properly understand functions and mechanisms of GSK-3, especially its role in glucose homeostasis via effects on glycogen synthase.

Resumo O diabetes mellitus (DM) é uma doença não transmissível em todo o mundo, na qual existe nível glicêmico persistentemente alto em relação à normalidade. O diabetes e a resistência à insulina são os principais responsáveis ​​pelas morbidades e mortalidades de humanos no mundo. Essa doença é regulada principalmente por várias enzimas e hormônios, entre os quais a glicogênio sintase quinase-3 (GSK-3) é uma enzima principal e a insulina é o principal hormônio que a regula. A GSK-3, que é a enzima-chave, normalmente mostra suas ações por vários mecanismos que incluem sua fosforilação, formação de complexos de proteínas e outras distribuições celulares e, portanto, controla e afeta diretamente a morfologia celular, seu crescimento, mobilidade e apoptose do célula. Perturbações na ação da enzima GSK-3 podem levar a várias condições de doença que incluem resistência à insulina que leva ao diabetes, doenças neurológicas como a doença de Alzheimer e câncer. As fluoroquinolonas são a classe mais comum de drogas que apresentam efeitos disglicêmicos por meio da interação com a enzima GSK-3. Portanto, é necessário hoje em dia compreender adequadamente as funções e mecanismos da GSK-3, principalmente seu papel na homeostase da glicose via efeitos na glicogênio sintase.

Glycogen synthase kinase-3 (GSK-3) a magic enzyme: it’s role in diabetes mellitus and glucose homeostasis, interactions with fluroquionlones. A mini-review / Glicogênio sintase quinase-3 (GSK-3), uma enzima mágica: seu papel no diabetes mellitus e na homeostase da glicose: interações com fluoroquinolonas. Uma mini-revisão

Diabetes mellitus (DM) is a non-communicable disease throughout the world in which there is persistently high blood glucose level from the normal range. The diabetes and insulin resistance are mainly responsible for the morbidities and mortalities of humans in the world. This disease is mainly regulated by various enzymes and hormones among which Glycogen synthase kinase-3 (GSK-3) is a principle enzyme and insulin is the key hormone regulating it. The GSK-3, that is the key enzyme is normally showing its actions by various mechanisms that include its phosphorylation, formation of protein complexes, and other cellular distribution and thus it control and directly affects cellular morphology, its growth, mobility and apoptosis of the cell. Disturbances in the action of GSK-3 enzyme may leads to various disease conditions that include insulin resistance leading to diabetes, neurological disease like Alzheimer’s disease and cancer. Fluoroquinolones are the most common class of drugs that shows dysglycemic effects via interacting with GSK-3 enzyme. Therefore, it is the need of the day to properly understand functions and mechanisms of GSK-3, especially its role in glucose homeostasis via effects on glycogen synthase.

O diabetes mellitus (DM) é uma doença não transmissível em todo o mundo, na qual existe nível glicêmico persistentemente alto em relação à normalidade. O diabetes e a resistência à insulina são os principais responsáveis pelas morbidades e mortalidades de humanos no mundo. Essa doença é regulada principalmente por várias enzimas e hormônios, entre os quais a glicogênio sintase quinase-3 (GSK-3) é uma enzima principal e a insulina é o principal hormônio que a regula. A GSK-3, que é a enzima-chave, normalmente mostra suas ações por vários mecanismos que incluem sua fosforilação, formação de complexos de proteínas e outras distribuições celulares e, portanto, controla e afeta diretamente a morfologia celular, seu crescimento, mobilidade e apoptose do célula. Perturbações na ação da enzima GSK-3 podem levar a várias condições de doença que incluem resistência à insulina que leva ao diabetes, doenças neurológicas como a doença de Alzheimer e câncer. As fluoroquinolonas são a classe mais comum de drogas que apresentam efeitos disglicêmicos por meio da interação com a enzima GSK-3. Portanto, é necessário hoje em dia compreender adequadamente as funções e mecanismos da GSK-3, principalmente seu papel na homeostase da glicose via efeitos na glicogênio sintase.

Glycogen synthase kinase-3 (GSK-3) a magic enzyme: its role in diabetes mellitus and glucose homeostasis, interactions with fluroquionlones. A mini-review

Abstract Diabetes mellitus (DM) is a non-communicable disease throughout the world in which there is persistently high blood glucose level from the normal range. The diabetes and insulin resistance are mainly responsible for the morbidities and mortalities of humans in the world. This disease is mainly regulated by various enzymes and hormones among which Glycogen synthase kinase-3 (GSK-3) is a principle enzyme and insulin is the key hormone regulating it. The GSK-3, that is the key enzyme is normally showing its actions by various mechanisms that include its phosphorylation, formation of protein complexes, and other cellular distribution and thus it control and directly affects cellular morphology, its growth, mobility and apoptosis of the cell. Disturbances in the action of GSK-3 enzyme may leads to various disease conditions that include insulin resistance leading to diabetes, neurological disease like Alzheimers disease and cancer. Fluoroquinolones are the most common class of drugs that shows dysglycemic effects via interacting with GSK-3 enzyme. Therefore, it is the need of the day to properly understand functions and mechanisms of GSK-3, especially its role in glucose homeostasis via effects on glycogen synthase.

Resumo O diabetes mellitus (DM) é uma doença não transmissível em todo o mundo, na qual existe nível glicêmico persistentemente alto em relação à normalidade. O diabetes e a resistência à insulina são os principais responsáveis pelas morbidades e mortalidades de humanos no mundo. Essa doença é regulada principalmente por várias enzimas e hormônios, entre os quais a glicogênio sintase quinase-3 (GSK-3) é uma enzima principal e a insulina é o principal hormônio que a regula. A GSK-3, que é a enzima-chave, normalmente mostra suas ações por vários mecanismos que incluem sua fosforilação, formação de complexos de proteínas e outras distribuições celulares e, portanto, controla e afeta diretamente a morfologia celular, seu crescimento, mobilidade e apoptose do célula. Perturbações na ação da enzima GSK-3 podem levar a várias condições de doença que incluem resistência à insulina que leva ao diabetes, doenças neurológicas como a doença de Alzheimer e câncer. As fluoroquinolonas são a classe mais comum de drogas que apresentam efeitos disglicêmicos por meio da interação com a enzima GSK-3. Portanto, é necessário hoje em dia compreender adequadamente as funções e mecanismos da GSK-3, principalmente seu papel na homeostase da glicose via efeitos na glicogênio sintase.

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.

Medicinal plants with antimalarial activities mediated via glycogen synthase kinase-3 beta (GSK3β) inhibition

@#Many of the therapeutic effects of plant extracts and bioactive compounds appear related to their immunomodulatory effects and impact on the host immune system. The immune response is desirable to mitigate established infections and, in the case of severe malaria, is a feasible approach to dealing with the overwhelming cytokine response. Glycogen synthase kinase-3 (GSK3), a Ser/Thr kinase that is a central regulator of the cytokine response, is a promising antimalarial drug target. In this review, we discussed our ongoing research projects, which include assessing the antimalarial activities of medicinal plants and their bioactive compounds, immunomodulatory activities mediated by GSK3, and the potential inflammatory pathway involved in malarial infection.