Study on the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis / 中国药房

OBJECTIVE To explore the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis （HF）. METHODS Intragastrical administration of carbon tetrachloride was given to induce HF model. By observing the pathological changes in liver tissue， mRNA and protein expressions of HF indexes [α-smooth muscle actin （α-SMA）， collagen type Ⅰ] and phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） pathway-related factors were detected， and the improvement effects and possible mechanism of low-dose， medium-dose and high-dose （50， 100， 200 mg/kg） of alcoholic extract from S. comosa on HF model rats were investigated. Drug-containing serum was prepared by intragastrical administration of alcoholic extract from S. comosa at a concentration of 1 800 mg/（kg·d） （calculated by the amount of raw material）. The effects of drug- containing serum of alcoholic extract from S. comosa on the expression of miRNA-21 were observed through the intervention of HSC-T6 cells with low， medium and high concentrations of drug-containing serum of alcoholic extract from S. comosa （diluted to 10%， 15%， 20%）. miRNA-21 mimics or inhibitors were used to transfect HSC-T6 cells， and the mRNA and protein expressions of factors related to the PI3K/Akt signaling pathway were detected. RESULTS The results of in vivo experiments showed that low， medium and high doses of alcoholic extract from S. comosa significantly ameliorated the histopathological changes in liver tissue of HF rats， and the percentage of collagen was significantly reduced （P＜0.01）； mRNA and protein expressions of the indicators related to HF as well as PI3K and Akt were significantly reduced （P＜0.01）， and mRNA and protein expressions of phosphatase and tensin homolog deleted on chromosome ten （PTEN） were increased in liver tissue of rats （P＜0.01）. The results of in vitro experiments showed that drug-containing serum of alcoholic extract from S. comosa significantly inhibited the expression of miRNA-21 at low， medium and high concentrations （P＜0.01）； whereas after transfection with miRNA-21 mimics， it was found that miRNA-21 mimics significantly increased mRNA and protein expressions of PI3K and Akt （P＜0.01）， while significantly decreased mRNA and protein expressions of PTEN （P＜0.01）； after transfection with miRNA-21 inhibitor， the changes of above indexes were opposite to the above results （P＜0.01）. CONCLUSIONS Alcoholic extracts of S. comosa may inhibit the PI3K/Akt signaling pathway by affecting the expression of miRNA-21， so as to achieve the effect of anti-hepatic fibrosis.

Ferulic acid inhibits the progression of T-cell acute lymphoblastic leukemia by regulating PTEN/PI3K/AKT signaling pathway / 局解手术学杂志

Objective To explore whether ferulic acid can inhibit the progression of T-cell acute lymphoblastic leukemia in vivo and in vitro by regulating PTEN/PI3K/AKT signaling pathway.Methods The T-cell acute lymphoblastic leukemia Jurkat cells were divided into the control group,the ferulic acid treatment group and the LY294002 treatment group for in vitro experiment.The cells in the control group were given normal culture;cells in the ferulic acid treatment group were given different concentrations(1.25,2.5,5,10,20,40,80,160 μmol/L)of ferulic acid,respectively,and the cell proliferation was detected by CCK-8 method,to screen the experimental concentration;cells in the LY294002 treatment group were given 50 μmol/L PI3K/AKT inhibitor LY294002.The cells proliferation,apoptosis and invasion were detected by clone formation assay,flow cytometry and Transwell assay.The relative expression levels of nuclear protein Ki67,proliferating cell nuclear antigen(PCNA),cleaved caspase-3,cleaved caspase-9,E-cadherin,N-cadherin,Vimentin,PTEN,p-PI3K,PI3K,p-AKT and AKT proteins were detected by Western blot.The nude mice models of transplanted tumors were constructed by 30 male BALB/c nude mice,and they were averagely divided into the normal group and the ferulic acid treatment group for in vivo experiment.The normal group was given normal saline by gavage,while the ferulic acid treatment group was given 75 mg/kg ferulic acid by gavage after inoculating Jurkat cells.The weight and volume changes of transplanted tumors were compared,and the levels of Ki67,cleaved caspase-3/caspase-3,E-cadherin,N-cadherin,PTEN,p-PI3K,PI3K,p-AKT and AKT in tumor tissues were detected.Results In vitro experiment,compared with the control group,the clone formation rate of cells,number of invasion cells,Ki67,PCNA,N-cadherin,Vimentin,p-PI3K/PI3K and p-AKT/AKT in the 5,10,20 μmol/L ferulic acid treatment group and the LY294002 treatment group were significantly decreased(P<0.05),while the apoptosis rate,cleaved caspase-3/caspase-3,cleaved caspase-9/caspase-9,E-cadherin and PTEN were significantly increased(P<0.05).In vivo experiment,compared with the normal group,the weight and volume of tumors were reduced in the ferulic acid treatment group,Ki67,N-cadherin,p-PI3K/PI3K and p-AKT/AKT in tumor tissues were significantly decreased,cleaved caspase-3/caspase-3,E-cadherin and PTEN were significantly increased,with statistically significant differences(P<0.05).Conclusion Ferulic acid can inhibit the proliferation and invasion of T-cell acute lymphoblastic leukemia Jurkat cells in vivo and in vitro,and induce apoptosis,its mechanism may be related to the regulation of PTEN/PI3K/AKT signaling pathway.

Expression and significance of PIK3CA,p-AKT and PTEN in sinonasal squamous cell carcinoma / 重庆医学

Objective To investigate the expression of PIK3CA,phosphorylated protein kinase B(p-AKT)and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in sinonasal squamous cell carcinoma(SNSCC).Methods The expressions of PIK3CA and PTEN in head and neck squamous cell carci-noma(HNSCC)were analyzed through the data set of HNSCC in the cancer genome map of UCSC Xena data-base.The immunohistochemical SP method was used to measure the expression of PIK3CA,p-AKT and PTEN in 43 cases of SNSCC tissues,20 cases of normal inferior concha tissues.The relationship between the expressions of PIK3CA,p-AKT and PTEN protein with the clinicopathological features and prognosis of the patients with SNSCC was analyzed.Results The results of bioinformatic analysis showed that PIK3CA mR-NA expression in HNSCC tissues was higher than that in paracancerous tissues(P＜0.01),while the PTEN mRNA expression was lower than that in paracancerous tissues(P＜0.05).The immunohistochemical detec-tion results showed that the positive expressions rates of PIK3CA and p-AKT proteins in normal nasal mucosa tissues were significantly lower than those in SNSCC tissues,while the positive expression rate of PTEN pro-tein in SNSCC tissues was significantly higher than that in normal inferior nasal concha mucosa tissues,and the differences were statistically significant(P＜0.01).The expressions of PIK3CA and p-AKT protein were related to the clinical stage,differentiation degree and primary site(P＜0.05),but were not related to age,gender,smoking and drinking(P＞0.05);the PTEN protein expression was not related with the clinical stage,differentiation degree,primary site,age,smoking and drinking(P＞0.05).The Spearman analysis showed that the expression of PIK3CA in SNSCC tissues was positively correlated with p-AKT protein ex-pression(r=0.664,P＜0.01),and PIK3CA was negatively correlated with PTEN protein(r=-0.414,P＜0.01).The expression of p-AKT was negatively correlated with PTEN protein(r=-0.453,P＜0.01).The Kaplan-Meier analysis showed that the median survival time of the patients with PIK3CA and p-AKT protein positive expression was shorter than that of the patients with negative expression(P＜0.01).There was no statistically significant difference in median survival between the patients with PTEN protein positive expres-sion and those with negative expression.Conclusion The overexpressions of PIK3CA and p-AKT accompa-nied by the loss of PTEN expression participate in the development and progression of SNSCC,moreover the PIK3CA and p-AKT expressions are related to the poor prognosis of the patients.

Effect of ganoderma spore on mitochondrial autophagy and apoptosis in testicular tissue of diabetic rats / 中国组织工程研究

BACKGROUND:As a common complication of diabetes mellitus,male reproductive disorders have received increasing attention in recent years.Ganoderma spore have hypoglycemic,antioxidant and anti-inflammatory effects,but the regulatory mechanism for diabetic testicular tissue has not been fully elucidated. OBJECTIVE:To investigate the effect of ganoderma spore on the PTEN-induced kinase 1/E3 ubiquitin ligase pathway and cell apoptosis in testicular tissue of diabetic rats. METHODS:Forty male Sprague-Dawley rats were randomly divided into normal group,high fat and high sugar group,diabetic group and ganoderma spore group,with 10 rats in each group.The latter three groups were given high fat/high sugar diet until the end of the experiment.After 1 month of high fat/high sugar diet,the diabetic and ganoderma spore groups were given intraperitoneal injection of streptozotocin(30 mg/kg per day)to establish type 2 diabetic rat models.After successful modeling,the ganoderma spore group was intragastrically given ganoderma spore(300 mg/kg per day),and the other groups were given the same amount of normal saline for continuous 12 weeks.The sperm number and morphology were detected.The histopathological changes of the testicle were observed.Serum testosterone and oxidative stress levels in testicular tissue were measured.The levels of PTEN-induced kinase 1,E3 ubiquitin ligase,and anti-nucleoporin p62 were analyzed by immunohistochemistry and the expression of PTEN-induced kinase 1,E3 ubiquitin ligase,anti-nucleoporin p62,programmed cell death-1,microtubule-associated protein light chain 3 Ⅱ/Ⅰ,caspase 3,cleaved-caspase 3 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the normal group and the high fat and high sugar group,the diabetic group had decreased sperm number(P＜0.01),increased sperm malformation rate(P＜0.01),and decreased serum testosterone level(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could increase the sperm number(P＜0.05),decrease the malformation rate(P＜0.01),and increase the serum testosterone level(P＜0.01).Compared with the normal group and the high fat and high sugar group,the malondialdehyde level in testis tissue was increased in the diabetic group(P＜0.01),while the levels of glutathione deoxidase and superoxide dismutase decreased(P＜0.01).Compared with the diabetic group,the malondialdehyde level in testis tissue was decreased in the ganoderma spore group(P＜0.01),and the levels of glutathione deoxidase and superoxide dismutase increased(P＜0.01).Immunohistochemical staining showed that compared with the normal group and the high fat and high sugar group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue were decreased in the diabetic group,while the positive expressions of anti-nucleoporin p62 were increased.Compared with the diabetic group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue e were increased in the ganoderma spore group,while the positive expression of anti-nucleoporin p62 was decreased.Western blot assay results indicated that compared to the normal group and the high fat and high sugar group,the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein were decreased in the diabetic group(P＜0.05 or P＜0.01),while the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3 were increased(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could elevate the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein(P＜0.05 or P＜0.01)as well as reduce the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3(P＜0.05 or P＜0.01).Overall,ganoderma spores may activate the PTEN-induced kinase 1/E3 ubiquitin ligase pathway to enhance autophagy in testicular tissue and reduce apoptosis in tissue cells,so as to protect testicular tissue.

Duhuo Jisheng Decoction treats gouty arthritis by inhibiting NLRP3 inflammasome activation / 中国组织工程研究

BACKGROUND:The activation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome has been found to be an important factor in the pathogenesis of gouty arthritis,and the activation of NLPR3 inflammasome can be effectively inhibited by regulating the PTEN-induced kinas 1(PINK1)/Parkin signaling pathway. OBJECTIVE:To investigate the effect of Duhuo Jisheng Decoction on the PINK1/Parkin/NLRP3 signaling pathway. METHODS:(1)Animal experiment:Male Sprague-Dawley rats were randomly divided into control group,model group,positive control group(Qiushuixian tablets 0.3 mg/kg),and low-,medium-,and high-dose Duhuo Jisheng Decoction groups(6.9,13.8,and 27.6 g/kg,respectively).A rat gouty arthritis model was constructed by injecting monosodium urate crystal suspension into the right hind ankle joint cavity of rats in all the groups except for the control group.(2)Cell experiment:Human monocytes(THP-1)were divided into blank control group,model group,Duhuo Jisheng Decoction-containing serum group,and PINK1 siRNA+Duhuo Jisheng Decoction-containing serum group.The cells in each group except the blank control group were stimulated with sodium urate to establish an in vitro gouty arthritis model.(3)The swelling degree,joint circumference,gait score,joint inflammation index and degree of pathological grading in the ankle joints of rats were observed.Enzyme-linked immunosorbent assay was used to detect the levels of uric acid,C-reactive protein,NLRP3,and interleukin 1β.Immunoblotting assay was used to detect the levels of proteins related to the PINK1/Parkin/NLRP3 pathway.Tetramethyl azolidine blue assay was used to detect cell activity.Immunofluorescent double staining was performed to detect the level of mitophagy.Immunofluorescence was used to detect the expression of NLRP3 and interleukin 1β. RESULTS AND CONCLUSION:(1)Compared with the control group,rats in the model group showed elevated ankle swelling and ankle circumference at 6-48 hours(P<0.05),elevated gait scores and joint inflammation indexes at 24 and 48 hours(P<0.05),elevated serum uric acid and C-reactive protein levels,degree of pathological classification,expression of PINK1,Parkin,and NLRP3 proteins,and interleukin 1β level in synovial tissues(P<0.05).Compared with the model group,the protein expression levels of PINK1 and Parkin in all the Duhuo Jisheng Decoction groups were elevated,while the levels of the other indexes were decreased(P<0.05).(2)Compared with the model group,NLRP3 activity,interleukin 1β level and mitochondrial outer membrane translocator protein 20 protein level in the Duhuo Jisheng Decoction-containing serum group were decreased(P<0.05),while the proliferation inhibition rate,PINK1,Parkin and LC3B protein level were increased(P<0.05).Compared with the model group,the mitochondrial autophagy level of cells in the Duhuo Jisheng Decoction-containing serum group was elevated(P<0.05),while NLRP3 and interleukin 1β protein levels were decreased(P<0.05).(3)Compared with the Duhuo Jisheng Decoction-containing serum group,the mitochondrial autophagy level of cells in the PINK1 siRNA+Duhuo Jisheng Decoction-containing serum group was decreased(P<0.05),and the expression levels of NLRP3 and interleukin 1β were elevated(P<0.05).To conclude,Duhuo Jisheng Decoction inhibits the activation of NLRP3 inflammasome by regulating the PINK1/Parkin signaling pathway, thereby exerting a therapeutic effect on gouty arthritis.

Levosimendan inhibits lipopolysaccharide-induced C2C12 cell apoptosis via the PTEN/Akt pathway / 中国医科大学学报

Objective To investigate whether levosimendan can inhibit the apoptosis of C2C12 cells induced by lipopolysaccharide(LPS)through the PTEN/Akt pathway.Methods C2C12 cells were randomly divided into four groups:blank control group,control group comprising cells treated with levosimendan only,LPS-treated group,and a group comprising cells pretreated with levosimendan for 24 h a subsequently treated with LPS for 48 h.The survival rate of C2C12 cells was determined via the CCK-8 method,and cell apoptosis was assessed via Hoechst 33342 staining.The mRNA and protein expression levels of PTEN/Akt pathway components were evaluated via RT-qPCR and Western blotting,respectively.C2C12 cells were also transfected with siRNA to knockdown the PTEN gene,and the effect on the protein expression of apoptotic pathway components was determined.Results Levosimendan increased the survival rate and decreased the apoptosis rate of C2C12 cells after LPS treatment.PTEN gene expression was inhibited by siRNA and the mRNA and protein levels of PTEN/Akt signaling pathway components changed correspondingly.Furthermore,the apoptosis rate of C2C12 cells decreased.Conclusion Levosimendan can inhibit LPS-induced C2C12 cell apoptosis via the PTEN/Akt pathway.

Effect of fenofibrate on the expression of miR-26a-5p/PTEN in retinal neurons of diabetic mice / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the protective effect of fenofibrate on diabetic retinal neurodegeneration and observe its effect on miR-26a-5p and its target gene PTEN in the retinal of diabetic mice.METHODS: Diabetic mice models were established and they were gavaged by fenofibrate. H&#x0026;#x0026; E staining and transmission electron microscopy were used to observe the impairments of retinal neurons. Real-time PCR was used to examine the expression of miR-26a-5p, and Western blotting was employed to measure the expression of phosphatase and tensin homologue(PTEN)in the retina of diabetic mice. The expression level of nuclear factor-κB(NF-κB), interleukin-1β(IL-1β)and the morphology of neural tissues were observed.RESULTS: When compared with the diabetic mice, fenofibrate significantly attenuated the damage to retinal ganglion cells and the atrophy of retinal nerve fiber layer. While the level of miR-26a-5p was increased and the levels of PTEN and inflammatory mediators were significantly decreased in the retina of fenofibrate treated diabetic mice, with significant statistical significance(P&#x0026;#x003C;0.05).CONCLUSIONS: Fenofibrate protects against diabetic retinal neurodegeneration by upregulating miR-26a-5p and inhibiting PTEN, attenuating the inflammatory response and alleviating retinal cell injury.

Mechanism of oxymatrine promoting the apoptosis of osteosarcoma MG63 cell line through mitophagy mediated by COX-2/PINK1/Parkin signaling pathway / 中国药房

OBJECTIVE To study the mechanism of oxymatrine inducing apoptosis of osteosarcoma MG63 cell line based on mitophagy mediated by cyclooxygenase-2 （COX-2）/PTEN-induced putative kinase-1 （PINK1）/Parkinson disease protein-2 （Parkin） signaling pathway. METHODS MG63 cells were treated with 2.0， 4.0， 8.0 mg/mL oxymatrine and 6 μmol/L 5-fluorouracil， then the apoptotic rate， the expression of apoptosis-related proteins [B-cell lymphoma-2 （Bcl-2）， Bcl-2 related X protein （Bax）]， the proportion of decrease in mitochondrial membrane potential， the level of mitophagy as well as the protein expressions of PINK1， Parkin， and microtubule-associated protein 1 light chain-3Ⅱ （LC3-Ⅱ） were detected. PINK1 small interfering RNA （siRNA） was adopted to intervene in the expression of PINK1， the cells were divided into control group， PINK1 siRNA group， oxymatrine group， and PINK1 siRNA+oxymatrine group； the protein expressions of PINK1， Parkin， and LC3-Ⅱ， the proportion of decrease in mitochondrial membrane potential （MMP） as well as apoptotic rate were detected. The lentivirus infection technique was used to overexpress COX-2， the cells were divided into control group， oxymatrine group， COX-2 group， and COX-2+oxymatrine group. The protein expressions of COX-2， PINK1， and Parkin， as well as the proportion of decrease in MMP were detected. RESULTS After being treated with oxymatrine， the apoptotic rate， the protein expressions of Bax， PINK1， Parkin， and LC3-Ⅱ， the level of mitophagy as well as the proportion of decrease in MMP were significantly increased （P＜0.05）， while the protein expression of Bcl-2 was significantly decreased （P＜0.05）. Compared with the oxymatrine group， the protein expressions of PINK1， Parkin， and LC3-Ⅱ， apoptotic rate and the proportion of decrease in MMP were significantly decreased in PINK1 siRNA+oxymatrine group （P＜0.05）. Compared with the oxymatrine group， the protein expression of COX-2 in the COX-2+oxymatrine group was increased significantly （P＜0.05）， while the protein expressions of PINK1 and Parkin as well as the proportion of 526087266@qq.com decrease in MMP were decreased significantly （P＜0.05）. CONCLUSIONS Oxymatrine can mediate the overactivity of mitophagy based on the PINK1/Parkin signaling pathway by inhibiting COX-2 expression， thus promoting the apoptosis of the MG63 osteosarcoma cell line.

Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity

Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

Knockdown of PTEN promotes colon cancer progression and induces M2 macrophage polarization in the colon cancer cell environment

Objective: This article aims to study the effect of phosphate and tension homolog deleted on chromosome ten (PTEN) knockdown on colon cancer progression and macrophage polarization in the cancer environment. Materials and Methods and Results: The expression of PTEN in colon cancer tissues and colon cancer cells was significantly lower than in precancerous tissues or CCD-18Co cells, and the decrease was most evident in SW620 cells. The expressions of phosphate (p)-p38, c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), B-cell lymphoma-2 (Bcl-2) protein in colon cancer tissues and cells were significantly higher than in precancerous tissues or CCD-18Co cells (P-values < 0.05). Bcl-2-associated X (Bax) and Caspase-3 expressions in colon cancer tissues and cells were significantly lower than in precancerous tissues or CCD-18Co cells (P-values < 0.05). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was applied to measure cell viability. Transwell evaluated the cell migration and invasion ability. Si-PTEN improved the proliferation, migration, and invasion of SW620 cells (P-values < 0.05). The expression levels of arginase-1 (Arg-1), CD163, CD206 in colon cancer tissues were significantly higher than in precancerous tissues (P-values < 0.05). The cell cycle, the number of M1 and M2 double-positive cells were assessed by flow cytometry. Si-PTEN reduced the expression of tumor necrosis factor-alpha (TNF-?), interleukin-1beta (IL-1?), and inducible nitric oxide synthase (iNOS), which upregulated the expression of Arg-1, CD206, CD163, p-p38, JNK, and AP-1 (P-values < 0.05). Conclusion: Si-PTEN promoted colon cancer progression and induced the polarization of M2 tumor-associated macrophages in the colon cancer cell environment.

Superexpressão Gênica PTEN em Tecidos Miocárdicos de Pacientes de Cirurgia de Revascularização Miocárdica / Overexpression of the PTEN Gene in Myocardial Tissues of Coronary Bypass Surgery Patients

Resumo Fundamento: A doença arterial coronariana é um distúrbio complexo que causa morte em todo o mundo. Um dos genes envolvidos no desenvolvimento dessa doença pode ser o PTEN. Objetivos: Nosso objetivo foi investigar a expressão gênica e proteica do PTEN em amostras de tecido e sangue retiradas de pacientes submetidos à cirurgia de revascularização miocárdica. Métodos: Foram realizados estudos moleculares no Centro de estudos do genoma humano e células-tronco da Universidade Erciyes (GENKOK). Amostras do apêndice atrial direito e de sangue foram coletadas da veia central de 22 pacientes submetidos à cirurgia de revascularização miocárdica antes de iniciar e terminar a circulação extracorpórea. A expressão do PTEN foi determinada usando PCR quantitativo em tempo real e análise de Western Blot. O nível de significância aceito foi de p<0,05. Resultados: Não houve diferença significativa na expressão gênica do PTEN em amostras de sangue coletadas antes e depois da circulação extracorpórea. Entretanto, foi observado um aumento substancial nos níveis de expressão gênica e proteica de P-PTEN e PTEN nas amostras de tecido. A expressão gênica miocárdica PTEN aumentou significativamente ao final da circulação extracorpórea. A expressão gênica do PTEN no período pós-circulação extracorpórea aumentou em comparação com o período pré-circulação extracorpórea, mas não foi um aumento significativo em comparação com sujeitos saudáveis do grupo de controle. Conclusão: Este estudo revelou pela primeira vez o papel do gene PTEN analisando a expressão de mRNA e de proteína em pacientes com revascularização miocárdica, que se manifesta tanto em tecido miocárdico quanto em amostras de sangue. O aumento dos níveis de PTEN pode ser um marcador no tecido miocárdico para pacientes com doença arterial coronariana.

Abstract Background: Coronary artery disease is a complex disorder that causes death worldwide. One of the genes involved in developing this disease may be PTEN. Objectives: This study aimed to investigate the PTEN gene and protein expression in tissue and blood samples taken from coronary bypass surgery patients. Methods: Molecular studies were performed at Erciyes University Genome and Stem Cell Center (GENKOK). Right atrial appendage and blood samples were taken from the central vein of 22 coronary bypass surgery patients before starting and ending cardiopulmonary bypass. PTEN expression was determined using quantitative real-time PCR and western blot analysis. The significance level was accepted as p<0.05. Results: There was no significant difference in the PTEN gene expression in blood samples taken before and after cardiopulmonary bypass. However, a substantial increase in both protein and gene expression levels of P-PTEN and PTEN was observed in the tissue samples. Myocardial expression of the PTEN gene was significantly increased at the end of the cardiopulmonary bypass. PTEN gene expression in the post-cardiopulmonary bypass period was increased when compared to the pre-bypass period, but it was insignificant when compared to healthy controls. Conclusion: This study first revealed the role of the PTEN gene by analyzing both mRNA and protein expression in coronary bypass patients, appearing in both myocardial tissue and blood samples. Increased levels of PTEN may be a marker in myocardial tissue for patients with coronary artery disease.

Mechanism of Guishao Yunpi Decoction in Prevention and Treatment of Mammary Gland Hyperplasia Based on PTEN/PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveStudy on the mechanism of Guishao Yunpi decoction in preventing and treating breast hyperplasia based on phosphatase and tensin homolog （PTEN）/ phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathway. MethodSeventy SPF-grade SD rats were randomly assigned into blank group （n=15） and modeling group （n=55）. The mammary gland hyperplasia model of liver stagnation and spleen deficiency was established by the comprehensive modeling method （hunger and satiety abnormality + tail stimulation + estradiol benzoate + progesterone）， and then 5 rats were randomly selected for model verification. The modeled rats were then randomly assigned into five groups： model group， positive control （4 mg·kg-1·d-1 tamoxifen） group， and high-， medium-， and low-dose （17.2， 8.6， 4.3 g·kg-1·d-1， respectively） Guishao Yunpi decoction groups， with 10 rats in each group. The rats in the blank group and model group were given 10 mL·kg-1·d-1 distilled water， and those in other groups were orally administrated with corresponding drugs. After 30 days of treatment， the general living conditions of rats were observed， and the thickness of breast tissue was measured by an ultrasonic diagnostic instrument. The open field test was carried out for behavioral evaluation. The levels of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X （Bax） in the breast tissue were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein levels of PTEN， PI3K， and Akt in the breast tissue were determined by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with those in the blank group， the rats in the model group were irritable， curled up in clusters， and showed positive behavior in the open field test. The modeling led to nipple swelling and increased the breast thickness （P<0.05）. Moreover， the modeling elevated the level of Bcl-2 and lowered that of Bax in the breast tissue （P<0.05）， down-regulated the mRNA and protein levels of PTEN， and up-regulated the mRNA and protein levels of PI3K and Akt （P<0.05）. Compared with the model group， drug administration relieved the general survival state， the degree of nipple swelling， and the positive behavior in the open field test and reduced the breast thickness （P<0.05）. In addition， drug administration reduced the level of Bcl-2 and increased that of Bax in the breast tissue （P<0.05）， up-regulated the mRNA and protein levels of PTEN， and down-regulated the mRNA and protein levels of PI3K and Akt （P<0.05）. ConclusionGuishao Yunpi decoction can improve the general living conditions and alleviate the mammary gland hyperplasia of rats with the syndrome of liver depression and spleen deficiency， which may be realized by the regulation of the PTEN/PI3K/Akt pathway.

Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy / 中国实验方剂学杂志

ObjectiveTo observe the effects of Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and their combination on slow transit constipation via PTEN-induced putative kinase 1 （PINK1）/Parkin pathway-mediated mitophagy. MethodFifty-six male SD rats were randomly assigned into normal group， model group， natural recovery group， Aurantii Fructus Immaturus group， Atractylodis Macrocephalae Rhizoma group， Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group， and mosapride group， with 8 rats in each group. Slow transit constipation model was established by gavage with loperamide （3 mg·kg-1·d-1） for 14 days in other groups except the normal group. After successful modeling， except that the model group was continuously induced by loperamide， the normal group and the natural recovery group were administrated with 0.9% normal saline by gavage， and the rats in the Aurantii Fructus Immaturus （1.35 g·kg-1·d-1） group， the Atractylodis Macrocephalae Rhizoma （2.7 g·kg-1·d-1） group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma （4.05 g·kg-1·d-1） group， and the mosapride （1.56 mg·kg-1·d-1） group were administrated with corresponding drugs by gavage for 7 days. The amount of feces， fecal water content， and intestinal propulsion rate of rats were determined. The pathological changes of the colon were evaluated by hematoxylin-eosin （HE） staining and Alcian blue-periodic acid-Schiff （AB-PAS） staining. The activity of respiratory chain complex and the ultrastructure of the colon tissue were determined by ultraviolet spectrophotometry and observed by transmission electron microscopy， respectively. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of PINK1， Parkin， and p62， and Western blot to determine the protein levels of microtubule-associated protein 1 light chain 3 （LC3）， PINK1， and Parkin. ResultCompared with the normal group， the model group and the natural recovery group showed decreases in the amount of feces， fecal water content， intestinal propulsion rate （P<0.05，P<0.01）， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ in the colon tissue （P<0.05，P<0.01）. Further， the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 were up-regulated （P<0.01） and the mRNA level of p62 was down-regulated in the model group （P<0.05） and the natural recovery group. Compared with the model group and the natural recovery group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group showed increased amount of feces， fecal water content， intestinal propulsion rate， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ （P<0.05，P<0.01）. Moreover， the combination meliorated the degree of mitochondrial swelling in the colon tissue， down-regulated the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 （P<0.05，P<0.01）， and up-regulated the mRNA level of p62 （P<0.05）. ConclusionAurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma， and their combination may remedy the colonic motility disorders in rats with slow transit constipation by blocking PINK1/Parkin signaling pathway to inhibit the excessive mitophagy in interstitial cells of Cajal in the colon tissue.

Mechanism of Cinnamaldehyde in Promoting Wound Healing in Diabetes Rats via PINK1/Parkin-mediated Mitochondrial Autophagy / 中国实验方剂学杂志

ObjectiveTo establish a rat model of diabetic wound by feeding on a high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin （STZ） and surgical preparation of full-thickness skin defects， observe the effect of cinnamaldehyde on the wound healing of diabetes rats， and explore the therapeutic mechanism of cinnamaldehyde in improving wound healing of diabetes rats based on the PTEN-induced putative kinase （PINK1）/Parkin pathway-mediated mitochondrial autophagy. MethodForty-eight male SD rats were randomly divided into blank group （n=12） and diabetes group （n=36）. The diabetes group was further randomly divided into model group， cinnamaldehyde group， and Beifuxin group， with 12 rats in each group. The blank group and the model group received routine disinfection with physiological saline after creating the wounds， while the cinnamaldehyde group received topical application of polyethylene glycol 400 （PEG 400） gel containing 4 μmol·L-1 cinnamaldehyde， and the Beifuxin group received topical application of Beifuxin gel. Dressings were changed once daily. The wound healing rate of each group was observed. On the 7th and 14th days after intervention， the wound tissues of the rats were collected. Hematoxylin and eosin （HE） staining was performed to observe the pathological changes in the local tissues. Immunohistochemistry （IHC） was used to detect the expression of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， vascular endothelial growth factor （VEGF）， and collagen fibers. Immunofluorescence （IF） and Real-time polymerase chain reaction （Real-time PCR） were used to detect the protein， and mRNA expression of PINK1， Parkin， microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. ResultAfter intraperitoneal injection of STZ， compared with the blank group， the random blood glucose values of rats in the diabetic group increased significantly （P<0.01）， all higher than 16.7 mmol·L-1， and persistently hyperglycemic for some time after modeling. Compared with the blank group， the model group showed poor growth and healing of granulation tissue in the wounds， and the wound healing rate decreased （P<0.01）. On the 7th day after intervention， the blank group had squamous epithelial coverage on the wounds. Compared with the blank group， the model group only had a small amount of scab at the wound edges， with a large number of infiltrating inflammatory cells in the wounds. The protein expression levels of IL-6 and TNF-α in the tissues increased （P<0.01）， and the protein and mRNA levels of PINK1， Parkin， and LC3Ⅱ decreased （P<0.01）. On the 14th day after the intervention， the granulation tissue in the wounds of the blank group was mature and well-healed. Compared with the blank group， the model group still had infiltrating inflammatory cells and red blood cell exudation. The protein expression levels of VEGF and collagen fibers in the tissues decreased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ increased （P<0.01）. Compared with the model group， the cinnamaldehyde group and the Beifuxin group showed better wound healing， with increased wound healing rates （P<0.01）. On the 7th day after intervention， the protein expression levels of IL-6 and TNF-α in the tissues decreased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ increased （P<0.01）. On the 14th day after intervention， the protein expression levels of VEGF and collagen fibers in the tissues increased （P<0.01）， and the protein and mRNA expression levels of PINK1， Parkin， and LC3Ⅱ decreased （P<0.01）. ConclusionCinnamaldehyde can promote the wound healing of diabetes rats by increasing the wound healing rate， reducing the levels of inflammatory factors IL-6 and TNF-α， and increasing the levels of VEGF and collagen fibers. Its mechanism may be related to the regulation of the PINK1/Parkin signaling pathway， activation of mitochondrial autophagy， inhibition of inflammatory responses， and promotion of angiogenesis and collagen synthesis， thereby promoting the wound healing of diabetes rats.

DJ-1 over-expression promotes proliferation, migration, invasion and epithelial-mesenchymal transformation of human gastric cancer MGC803 cells through PTEN/Akt pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨DJ-1基因过表达对人胃癌MGC803细胞增殖、迁移、侵袭与上皮间质转化（EMT）的影响及其机制。方法：利用基因转染技术构建DJ-1基因过表达MGC803细胞，实验分为MGC803、空载体和DJ-1过表达组。采用MTT、平板克隆形成、细胞划痕和Transwell实验分别检测DJ-1过表达对MGC803细胞增殖、克隆形成、迁移与侵袭的影响；qPCR和WB法检测DJ-1过表达对各组细胞DJ-1、PTEN、Akt、p-Akt、Snail、vimentin、E-cadherin、MMP-9与TIMP-3表达的影响，相差显微镜下观察MGC803细胞形态学的变化。裸鼠荷瘤实验检测DJ-1过表达对MGC803细胞移植瘤体内生长的影响。结果：成功构建DJ-1基因稳定过表达的MGC803细胞。与MGC803组和空载体组比较，DJ-1过表达组细胞的增殖能力与克隆形成数均显著增加（均P<0.05），细胞迁移距离明显增加、划痕距离明显缩短（均P<0.05），迁移与侵袭细胞数显著增多（均P<0.05），DJ-1 mRNA与蛋白表达明显上调、PTEN mRNA与蛋白表达下调（均P<0.05），Akt总蛋白各组比较无明显差异（均P>0.05），p-Akt蛋白表达明显上调（P<0.05），Snail、vimentin与MMP-9表达上调、E-cadherin与TIMP-3表达下调（均P<0.05）。相差显微镜下见长梭形细胞数目增多，圆形与椭圆形细胞减少，异型性更为明显。荷瘤裸鼠体内实验结果表明，与MGC803组相比较，DJ-1过表达组MGC803细胞移植瘤生长速度明显加快、移植瘤质量显著增加（均P<0.05）。结论：DJ-1过表达可通过PTEN/Akt通路在体内外抑制MGC803细胞的增殖、迁移、侵袭与EMT。

Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway / 中国中药杂志

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Effect and Mechanism of Atorvastatin on Reversing Drug Resistance in Leukemia by Regulating Glycolysis through PTEN/mTOR Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM.@*METHODS@#HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected.@*RESULTS@#CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance.@*CONCLUSION@#Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment / 中国结合医学杂志

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

Effect on Danggui Shaoyao Powder on mitophagy in rat model of Alzheimer's disease based on PINK1-Parkin pathway / 中国中药杂志

This study investigated the mechanism of Danggui Shaoyao Powder(DSP) against mitophagy in rat model of Alzheimer's disease(AD) induced by streptozotocin(STZ) based on PTEN induced putative kinase 1(PINK1)-Parkin signaling pathway. The AD rat model was established by injecting STZ into the lateral ventricle, and the rats were divided into normal group, model group, DSP low-dose group(12 g·kg~(-1)·d~(-1)), DSP medium-dose group(24 g·kg~(-1)·d~(-1)), and DSP high-dose group(36 g·kg~(-1)·d~(-1)). Morris water maze test was used to detect the learning and memory function of the rats, and transmission electron microscopy and immunofluorescence were employed to detect mitophagy. The protein expression levels of PINK1, Parkin, LC3BⅠ/LC3BⅡ, and p62 were assayed by Western blot. Compared with the normal group, the model group showed a significant decrease in the learning and memory function(P<0.01), reduced protein expression of PINK1 and Parkin(P<0.05), increased protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05), and decreased occurrence of mitophagy(P<0.01). Compared with the model group, the DSP medium-and high-dose groups notably improved the learning and memory ability of AD rats, which mainly manifested as shortened escape latency, leng-thened time in target quadrants and elevated number of crossing the platform(P<0.05 or P<0.01), remarkably activated mitophagy(P<0.05), up-regulated the protein expression of PINK1 and Parkin, and down-regulated the protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05 or P<0.01). These results demonstrated that DSP might promote mitophagy mediated by PINK1-Parkin pathway to remove damaged mitochondria and improve mitochondrial function, thereby exerting a neuroprotective effect.