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ObjectiveTo explore the effects of Bushen Jianpi prescription on the autophagy and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway in the patients with aplastic anemia （AA）. MethodA total of 30 AA patients admitted to Xiyuan Hospital and 6 healthy donors who were prepared to undergo peripheral blood hematopoietic stem cell transplantation in 304 Hospital from September 2020 to August 2021 were enrolled and assigned into an AA group and a control group. The AA group was treated with Bushen Jianpi prescription combined with cyclosporin A （CsA） and androgen for 3 months. The mononuclear cells from bone marrow in the AA group before and after treatment and the peripheral blood of the control group were collected. Transmission electron microscopy was then employed to detect autophagosomes. Western blotting was employed to determine the protein levels of microtuble-associated protein 1 light chain 3 （LC3）Ⅰ， LC3Ⅱ， mTOR， phosphorylated （p）-mTOR， Akt， p-Akt， PI3K， and p-PI3K， and real-time polymerase chain reaction （PCR） to determine the mRNA levels of LC3， mTOR， Akt， and PI3K. ResultIn the AA group， the treatment was completed in 29 patients， and the total response rate was 51.72% （15/29）. ① The AA group showed lower levels of white blood cell （WBC）， hemoglobin （HGB）， platelet （PLT）， and absolute neutrophil count （ANC） in the peripheral blood （P<0.01） and lower number of intracellular autophagosomes than the control group before treatment. Moreover， the AA group showed lower mRNA level of LC3 （P<0.01） and protein levels of LC3Ⅰ and LC3Ⅱ （P<0.01） and higher mRNA levels of mTOR， Akt， and PI3Kα （P<0.01） and protein levels of Akt， p-Akt， PI3K， p-PI3K， mTOR， and p-mTOR （P<0.01） than the control group. ② In AA group， the levels of HGB and PLT elevated （P<0.05） and the number of intracellular autophagosomes increased after treatment compared with those before treatment. Moreover， the mRNA level of LC3 and the protein levels of LC3Ⅰ and LC3Ⅱ were up-regulated （P<0.01）， the mRNA levels of mTOR， Akt， and PI3Kα （P<0.01） and the protein levels of Akt， p-PI3K （P<0.01）， p-Akt， PI3K， mTOR， p-mTOR （P<0.05） were down-regulated after treatment. ConclusionAA patients show lower autophagy levels， while Bushen Jianpi prescription can effectively improve the autophagy level and down-regulated the expression of PI3K/Akt/mTOR signaling pathway in AA patients.
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ObjectiveTo observe the effects of Wendantang on the expression of inflammatory cytokines， autophagy markers， and key molecules of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway in the adipocytes of the rat model of obesity （syndrome of phlegm-dampness） and to explore the material basis of inflammation in obesity （syndrome of phlegm-dampness） and the underlying mechanism of Wendantang intervention. MethodA total of 126 SD rats were randomized into 2 groups： 16 rats in the blank group and 110 rats in the modeling group. The blank group was fed with a basic diet while the modeling group with a high-fat diet to establish the animal model of obesity （syndrome of phlegm-dampness） for 8 weeks. After successful modeling， 48 obese rats were selected according to their body mass and randomized into a model control group， an orlistat （ORLI， 32.40 mg·kg-1） group， a rapamycin （RAPA， 2 mg·kg-1） group， and low-， medium-， and high-dose （4.45， 8.90， 17.80 g·kg-1， respectively） Wendantang groups， with 8 rats in each group. In addition， 8 rats were randomly selected from the blank group to be set as the normal control group. The corresponding agents in each group were administrated by gavage and the model and control groups were administrated with equal amounts of distilled water once daily for 6 weeks. The body mass， Lee's index， body fat ratio， and obesity rate were measured or calculated. The expression of UNC51-like kinase-1 （ULK1）， Beclin1， human autophagy-related protein 5 （Atg5）， p62， and microtubule-associated protein 1 light chain 3 （LC3） Ⅰ/Ⅱ （markers of autophagy in adipocytes） was detected by the immunohistochemical two-step method. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the expression of tumor necrosis factor （TNF）-α， interleukin-6 （IL-6）， IL-1β， monocyte chemotactic protein-1 （MCP-1）， IL-4， IL-10， IL-13， and transforming growth factor （TGF）-β in adipocytes. Western blot was employed to measure the protein levels of classⅠ-PI3K， phosphatidylinositol triphosphate （PIP3）， Akt， mTORC1， ULK1， TSC1， and TSC2 in adipocytes. ResultCompared with the blank group， the modeling group showed increased body mass and Lee's index （P<0.01）， the obesity rate >20%， and phlegm-dampness syndrome manifestations such as physical obesity， decreased mobility， decreased appetite， lusterless and tight fur， loose stools， decreased responsiveness to the outside world， and decreased water intake. Compared with the normal control group， the model control group showed increased body mass， Lee's index， body fat ratio， adipocyte autophagy marker expression， pro- and anti-inflammatory cytokine levels （P<0.05， P<0.01）， down-regulated protein levels of classⅠ-PI3K， PIP3， Akt， mTORC1， TSC1， and TSC2 （P<0.01）， and up-regulated protein level of ULK1 （P<0.01）. The intervention groups showed lower body mass， body fat ratio， adipocyte autophagy marker protein expression， and protein levels of TNF-α， IL-6， IL-1β， MCP-1， IL-4， and IL-13 than the model control group （P<0.05， P<0.01）. Moreover， the RAPA and Wendantang （medium and high dose） groups showed lowered levels of IL-10 and TGF-β （P<0.01）， and the ORLI group showed down-regulated expression of TGF-β （P<0.01）. The expression of key molecules of the signaling pathway was up-regulated （P<0.05， P<0.01） while that of ULK1 was down-regulated （P<0.01） in all the intervention groups. Compared with the RAPA group， the Wendantang groups showed up-regulated expression of all autophagy marker proteins in adipocytes （P<0.01）. In addition， the low-dose Wendantang group showed elevated levels of inflammatory cytokines （except TNF-α） （P<0.05， P<0.01） and down-regulated expression of all key molecules of the signaling pathway （P<0.05， P<0.01）. The levels of inflammatory cytokines （except IL-16， MCP-1， and IL-10） were elevated in the medium-dose Wendantang group （P<0.05， P<0.01）. The expression of key molecules except PI3K of the signaling pathway was down-regulated in the medium- and high-dose Wendantang groups （P<0.05， P<0.01）. Compared with the ORLI group， low- and medium-dose Wendantang groups showed up-regulated expression of autophagy markers in adipocytes （P<0.01）， and the low-dose group showed elevated levels of inflammatory cytokines （IL-6， IL-4， and TGF-β） （P<0.01） and down-regulated expression of all key molecules of the signaling pathway （P<0.01）. The medium-dose Wendantang group showed up-regulated expression of IL-4 （P<0.01） and down-regulated expression of key molecules except PI3K of the signaling pathway （P<0.05， P<0.01）. The high-dose Wendantang group showed increased body mass， up-regulated expression levels of autophagy markers （ULK1， LC3 Ⅰ/Ⅱ） （P<0.05， P<0.01）， down-regulated expression of PIP3， mTORC1， and TSC1 （P<0.05， P<0.01）， and lowered levels of Beclin1， Atg5， TNF-α， and IL-13 （P<0.05， P<0.01）. ConclusionThe inflammation in obesity （syndrome of phlegm-dampness） is closely associated with the PI3K/Akt/mTOR pathway-mediated adipocyte autophagy. Wendantang can treat the chronic inflammation in obese rats with the syndrome of phlegm-dampness by regulating this signaling pathway and thus improve adipocyte autophagy.
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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.
Subject(s)
Female , Animals , Mice , Humans , Proto-Oncogene Proteins c-akt/genetics , Choristoma , Endometriosis/genetics , TOR Serine-Threonine Kinases/genetics , RNA, Messenger , Signal Transduction , Body Weight , Mammals , PTEN Phosphohydrolase/geneticsABSTRACT
ObjectiveTo observe the effect of Zhuluan decoction on the ovarian reserve function of rats with cyclophosphamide-induced premature ovarian insufficiency， and explore the protective mechanism of Zhuluan decoction in the rat model of premature ovarian insufficiency based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty female SD rats were randomly divided into normal group （n=10） and model group （n=50）. The model group was given intraperitoneal injection of cyclophosphamide （50 mg·kg-1 loading dose on the 1st day+8 mg·kg-1 low-dose maintenance on the 2nd–15th days）. After successfully modeling， the rats were randomly divided into model group， positive drug （progynova） group （0.1 mg·kg-1·d-1）， and low-， medium-， and high-dose Zhuluan decoction groups （14， 28， 56 g·kg-1·d-1 ）， with 10 rats in each group. The model group and the normal group were given equal volume of distilled water by gavage， once a day， continuous administration for 21 d. The estrous cycle and body weight of rats in each group were detected， and the ovarian organ index and uterine organ index were calculated. The ovarian tissue pathology and ovarian follicle counts at all levels were determined by hematoxylin-eosin （HE） staining. The content of the serum antimullerian hormone （AMH）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and inhibin-B （INH-B） of rats was determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of PI3K， Akt， mTOR in the rat ovarian tissue were determined by Western blot. The microtubule-associated protein 1 light chain 3B （LC3B） protein expression in the rat ovarian tissue was determined by immunohistochemistry. ResultAs compared with the blank group， the estrous cycle of rats in the model group was disordered， the body weight， ovarian organ index， and uterine organ index decreased， the number of primordial follicles decreased， and the number of secondary follicles and atretic follicles increased. In the model group， FSH increased （P<0.01）， LH increased （P<0.05）， AMH level decreased （P<0.05）， the protein expression levels of PI3K， Akt， and mTOR in the ovarian tissue decreased （P<0.01）， and the protein expression level of LC3B increased significantly （P<0.01）. As compared with the model group， the above indexes were improved in the progynova group and different doses of Zhuluan decoction groups， the content of AMH increased （P<0.05）， and FSH decreased （P<0.05）. In the progynova group and different doses of Zhuluan decoction groups， the protein expression level of LC3B decreased obviously （P<0.01）， and the protein expression levels of PI3K， Akt， and mTOR all showed an increasing trend. Moreover， there was a statistically significant difference in the progynova group and low- and medium-dose Zhuluan decoction groups （P<0.05）. ConclusionZhuluan decoction may inhibit the occurrence of excessive autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR pathway， thereby reversing the effect of modeling on ovarian reserve in rats.
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Osteoporosis （OP）， a common systemic skeletal disease in the elderly， is characterised by bone loss and bone microstructural degeneration. Its clinical manifestations include increased bone fragility and bone pain. Furthermore， OP increases the risk of fracture due to the high bone fragility， which leads to lifelong disability or death， imposing a heavy economic and psychological burden on the patients and their families. The pathogenesis of OP is extremely complex and associated with a variety of factors such as proliferation and differentiation of osteoblasts， impairment of osteoclast activity and function， and abnormalities in autophagy activation. Recent studies have found that mammalian target of rapamycin （mTOR） signaing pathway is involved in the regulation of bone homeostasis， which can promote bone formation and improve bone metabolism and bone microstructure by regulating osteoblast proliferation and differentiation and osteoclast function and activating cellular autophagy， thus playing a crucial role in the prevention and treatment of OP. The prevention and treatment of OP with Chinese medicine has a long history， clear efficacy， multiple targets of action， low adverse effects， and wide medicine sources. Therefore， this paper briefly describes the role of mTOR signaling pathway in the development of OP by reviewing the latest research reports and summarizes in detail the latest research results on the treatment of OP with Chinese medicine extracts and prescriptions via the mTOR signaling pathway. This review aims to provide a basis for the in-depth research on the relationship between mTOR signaling pathway and OP and the clinical application of traditional Chinese medicine in the prevention and treatment of OP.
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@#Objective To study the effect of Tangeretin on non-small cell lung cancer (NSCLC) and the tumor stemness, and to find the molecular mechanism of its effect. Methods We used cell counting and cell cloning experiments to study the effect of Tangeretin on the proliferation of NSCLC cells in vitro. The effect of Tangeretin on the invasion of NSCLC cells was detected by transwell assay. We detected the effect of Tangeretin on the proliferation of NSCLC cells in vivo by nude mouse tumor-bearing experiment. The effect of Tangeretin on tumor stemness of NSCLC cells was detected by self-renew assay, and CD133 and Nanog protein expressions. The expressions of PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blotting (WB). Results Tangeretin had a good inhibitory effect on the proliferation of NSCLC cells in vivo and in vitro. Cell counting experiment, clonal formation experiment and nude mouse tumor-bearing experiment showed that Tangeretin could inhibit the proliferation activity, clonal formation ability, and tumor size of NSCLC cells in vivo. Self-renew experiments showed that Tangeretin could inhibit the self-renew ability of NSCLC cells. WB experiments showed that Tangeretin inhibited the expressions of tumor stemness markers CD133 and Nanog in NSCLC cells. Tangeretin could inhibit the activation of PI3K/AKT/mTOR signaling pathway-related proteins in NSCLC cells, and the activation of PI3K/AKT/mTOR signaling pathway could partially remit the inhibitory effect of Tangeretin on tumor stemness of NSCLC cells. Conclusion Tangeretin can inhibit the tumor stemness of NSCLC cells, which may be related to the regulation of PI3K/AKT/mTOR signaling pathway.
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ABSTRACT Background: While sarcopenia is an important clinical finding in individuals diagnosed with chronic heart failure (CHF), efforts to identify a reliable biomarker capable of predicting the overall muscular and functional decline in CHF patients have been unsuccessful to date. Objectives: The objectives of this study were to study the diagnostic utility of MicroRNA (miRNA)-1-3p as a predictor of sarcopenia status in individuals diagnosed with CHF. Methods: In total, 80 individuals with heart failure exhibiting a left ventricular ejection fraction < 50% were enrolled in this study. All patients were analyzed to assess miR-1-3p expression levels, with body composition being evaluated through dual-energy X-ray absorptiometry and sarcopenia being defined based on the sum of appendicular lean muscle mass (ALM) divided by height in meters squared and handgrip strength (HGS). In addition, the activation of the Akt/mTOR signaling pathway was evaluated in these individuals. Results: In total, 40 of the enrolled patients (50%) exhibited sarcopenia. Sarcopenic patients presented with increased miR-1-3p expression levels as compared to non-sarcopenic individuals (1.69 ± 0.132 vs. 1.22 ± 0.106; p < 0.05). With respect to sarcopenic indices, appendicular skeletal mass index was most strongly correlated with miR-1-3p expression, which was also strongly correlated with HGS. High levels of Akt/mTOR signaling pathway components were expressed in sarcopenic individuals, highlighting a significant relationship between miR-1-3p activity and signaling through this pathway. Moreover, miR-1-3p was identified as a specific marker for sarcopenia in individuals with CHF. Conclusion: These results suggest that circulating miR-1-3p levels are related to Akt/mTOR pathway activation and can offer valuable insight into the overall physical capacity and muscular integrity of CHF patients as a predictor of sarcopenia.
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ObjectiveTo investigate the effect of betulinic acid （BA） on apoptosis and autophagy of human colorectal cancer SW620 cells and the regulatory role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodCell viability was detected by methyl thiazolyl tetrazolium （MTT） colorimetry to determine the optimal administration time and dosage for subsequent experiments. Four groups were designed， including blank group and low-， medium-， and high-dose BA groups. Hematoxylin-eosin （HE） staining was conducted for the observation of SW620 cell morphology， and annexin-V/propidium iodide double staining for the determination of apoptosis rate in SW620 cells. Hoechst33258 staining and MDC staining were used for the observation of apoptosis and autophagy， respectively. Western blotting was employed to determine the protein levels of B-cell lymphoma/leukemia-2（Bcl-2）-associated X protein （Bax）， aspartate proteolytic enzyme-9 （Caspase-9）， activated aspartate proteolytic enzyme-3 （cleaved Caspase-3）， microtubule-associated protein 1 light chain 3 （LC3）， the mammalian homolog of yeast Atg6 （Beclin-1）， p62， phosphorylated PI3K （p-PI3K）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） in SW620 cells. ResultBA inhibited the activity of SW620， HT29， and HCT116 cells in a concentration- and time-dependent manner. The cells treated with BA for 48 h had lower viability than those treated for 24 h （P<0.05， P<0.01）. The half maximal inhibitory concentration (IC50） value of BA at the time point of 48 h was also lower than that at the time point of 24 h （P<0.01）， and that for SW620 cells was the minimum. BA induced the apoptosis in a concentration-dependent manner and increased the autophagosomes. Compared with the blank group， BA increased the apoptosis rate （P<0.01）， up-regulated the protein levels of Bax， Caspase-9， cleaved Caspase-3， and LC3 Ⅱ （P<0.05， P<0.01）， and down-regulated the protein levels of p62， p-Akt， p-PI3K， and p-mTOR （P<0.01）. Additionally， medium- and high-dose BA up-regulated the protein level of beclin-1 （P<0.01）. ConclusionBA may inhibit the activity of SW620 cells by hindering the PI3K/Akt/mTOR signaling pathway to induce cell apoptosis and autophagy.
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ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.
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ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.
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OBJECTIVE：To study the repa ir effect and mechanism of Taohong siwu decoction on rotator cuff injury in rabbits. METHODS：New Zealand rabbits were randomly divided into blank control group ，model group ，Taohong siwu decoction low-dose，medium-dose and high-dose groups （2.75，5.5，11 g/kg），and Taohong siwu decoction+LY 294002 group [ 5.5 g/kg Taohong siwu decoction+ 6.4 μg/kg LY294002（pathway inhibitor ）]，with 11 rabbits in each group. Except for blank control group ， other groups underwent right subscapularis muscle detachment to establish rotator cuff injury model. After modeling ，blank control group and model group were given normal saline intragastrically ；Taohong siwu decoction groups were given relevant medicine intragastrically；Taohong siwu decoction+LY 294002 group was given Taohong siwu decoction intragastrically ，and then injected with LY 294002 at the ear edge ，once a day ，for 12 weeks. After last intragastric administration （injection），the pathological changes of tendon-bone interface was observed ；the levels of TNF-α，IL-10 and IL- 6 in serum were detected ；mRNA and protein expressions of PI 3K，Akt and mTOR in tendon-bone interface were detected ；the expression of autophagy related protein （Beclin1， LC3Ⅱ）were detected. RESULTS ：Compared with blank control group ，the tendon-bone interface was uneven and the intima was swollen in model group. Serum levels of TNF-α and IL-6，mRNA and protein expressions of PI 3K，Akt and mTOR in tendon-bone interface were increased significantly （P＜0.05 or P＜0.01），while the level of IL- 10 and protein expression of Beclin 1 and LC 3Ⅱ were decreased significantly （P＜0.01）. Compared with model group ，the tendon-bone interface of rabbits in Taohong siwu decoction low-dose and medium-dose groups still had certain intimal damage ，while the tendon-bone interface of rabbits in high-dose group was smooth and flat without an obvious intimal tear ；the levels of most indexes in serum and tendon-bone interface of rabbits were significantly reversed in each dose group （P＜0.05 or P＜0.01）. Swollen tendon-bone interface and obvious intimal tear were obse rved in Taohong siwu decoction + LY group；compared with model group ，there w as no significant difference in above indexes of serum and tendon-bone interface （P＞0.05）. CONCLUSIONS ：Taohong siwu decoction may repair the rotator cuff injury of rabbits ，the mechanism of which may be associated with inhibiting PI3K/Akt/mTOR signaling pathway，activating autophagy and inhibiting inflammatory response.
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OBJECTIVE：To study the reversal effect of quercetin on human cervical squamous carcinoma cisplatin-resistant cell line SiHa/DDP. METHODS ：The drug resistance index of cisplatin to SiHa/DDP cells ，and the reversal resistance multiple of quercetin to SiHa/DDP cells were determined. The effects of quercetin （0.005 μg/mL），cisplatin（2.5 μg/mL），cisplatin combined with quercetin （2.5 μg/mL cisplatin+0.005 μg/mL quercetin），quercetin combined with pathway inhibitor（0.005 μg/mL quercetin+ 20 nmol/L rapamycin ）on the apoptotic rate of SiHa/DDP cells were investigated ，as well as its effects on the expression of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian rapamycin target protein （mTOR） signaling pathway related proteins （PI3K，Akt，mTOR，P-gp，p70S6K）. RESULTS ：The resistance index of cisplatin to SiHa/DDP cells was 5.19， and reversal resistance multiple of quercetin to SiHa/DDP cells was 4.00. Compared with cisplatin alone and quercetin alone ， cisplatin combined with quercetin ，quercetin combined with rapamycin could significantly increase the apoptotic rate of SiHa/DDP cells（P＜0.05），while decreased the phosphorylation of Akt ，mTOR and p 70S6K protein as well as the expression of P-gp protein （P＜0.05）. CONCLUSIONS ：Quercetin can effectively reverse drug resistance of SiHa/DDP cells to cisplatin ，which may be associated with inhibiting the expression of the protein related to PI 3K/Akt/mTOR signaling pathway.
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OBJECTIVE：To study the improvement eff ects and p otential mechanism of chelidonine on CCl 4-induced hepatic fibrosis model rats. METHODS ：According to the random number table method ，a total of 48 rats were randomly divided into normal control group ，model group ，chelidonine low-dose ，middle-dose and high-dose groups （0.125，0.25，0.50 mg/kg），positive control group （Liver-protecting tablet ，0.42 g/kg），with 8 rats in each group. Except for normal control group ，other groups were given CCl 4-olive oil solution intraperitoneally for 8 weeks to induce hepatic fibrosis model. From the fifth week of modeling ， normal control group and model group were given water intragastrically ；administration groups were given relevant medicine intragastrically，once a day ，for consecutive 10 weeks. After last intragastric administration ，hepatic index of rats was calculated. The levels of aspartate aminotransferase （AST），alanine aminotransferase （ALT）and hyaluronic acid （HA）in serum and the level of hydroxyproline （Hyp）in liver tissue were determined. The staining of collagen fibrin in rat liver was observed. The protein expression of α-smooth muscle actin （α-SMA），microtubule-associated protein 1 light chain 3（LC3）and p 62 as well as the phosphorylation level of phosphoinositide 3 kinase（PI3K），protein kinase B （Akt）and mammalian target of rapamycin （mTOR）in liver tissue were determined ；mRNA expression corresponding to above protein were also determined. RESULTS ：Compared with normal control group ，the hepatic index ，the serum levels of AST ，ALT，HA and Hyp ，the percentage of positive staining area for collagen fibrin ，the mRNA and protein expression of α-SMA and LC 3- Ⅱ were increased significantly （P＜0.05）. Protein expression of p 62，phosphorylation levels of PI 3K，Akt and mTOR as well as mRNA expression of p 62，PI3K，Akt and mTOR were significantly down-regulated （P＜0.05）. Compared with model group ，phosphorylation levels of PI 3K and mTOR were decreased significantly in chelidonine low-dose group （P＜0.05）. The changes of above indexes in chelidonine middle-dose and high-dose groups （except for liver index ， HA level in middle-dose group ） were reversed significantly （P＜0.05）. CONCLUSIONS：Chelidonine can attenuate CCl 4-induced liver fibrosis in rats ；the mechanism of it may be associated with activating PI 3K/Akt/mTOR signaling pathway and inhibiting autophagy.
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Objective:To investigate the effect of Guizhi Fulingwan on ovulation dysfunction in rats with polycystic ovary syndrome with insulin resistance （PCOS-IR） induced by letrazole combined with high fat emulsion. Method:A total of 72 female SD rats were randomly divided into control group， model group， metformin group and Guizhi Fulingwan low， medium and high dose groups， with 12 rats in each group. Except for control group， rats were given letrozole 0.001g·kg<sup>-1</sup> combined with high-fat emulsion 15 mL·kg<sup>-1</sup> for 21 consecutive days to establish model of PCOS-IR. Guizhi Fulingwan low， medium and high-dose groups were administrated with Guizhi Fulingwan 0.31， 0.62， 1.24 g·kg<sup>-1</sup> respectively， metformin group was administrated with metformin 0.27 g·kg<sup>-1</sup>， control group and model group were administrated with 12 mL·kg<sup>-1</sup> of normal saline daily for 30 days. Hematoxylin-eosin（HE） staining was used to observe ovarian tissue pathology morphology， and enzyme-linked immunoassay method （ELISA） was used to detect serum follicle stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， fasting insulin （FINS） level，and LH/FSH and insulin resistance index （HOMA-IR） were calculated． Western blot was used to detect the expression levels of autophagy key molecular Atg6 yeast homologue （Beclin-1）， autophagy related gene 5（Atg5）， microtubule associated protein light chain 3 （LC3） Ⅱ proteins in the phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein （PI3K/Akt/mTOR） signaling pathway and autophagy related indicators in rat ovarian tissue. Beclin-1 and LC3Ⅱ protein expressions were detected by immunohistochemistry （IHC）. Result:Compared with control group， the thickness of follicles and follicular granulosa cells in the ovary of the model group also decreased， and the number of corpus luteum significantly decreased， while the white membrane thickness of the ovary increased， and the number of atresia follicles and cystic dilatation follicles increased significantly. Serum T， LH， LH/FSH， FINS， FINS， HOMA-IR were significantly increased （<italic>P</italic><0.01）. Phosphorylated （p） -PI3K， p-Akt， and p-mTOR proteins in ovarian tissue were all decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The relative expression levels of autophagy-related protein LC3Ⅱ and Beclin-1 were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， the number of follicles in the low， medium and high dose Guizhi Fulingwan group and the metformin group decreased， the number of follicles in atresia and atresia increased， and the follicular granulosa cell layer thickness increased. Serum T， LH， LH/FSH， FINS and HOMA-IR of Guizhi Fulingwan group were significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， and serum FINS and HOMA-IR of metformin group were significantly decreased （<italic>P</italic><0.01）. The expressions of p-PI3K， p-Akt， and p-mTOR proteins were increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of LC3Ⅱ， Atg5 and Beclin-1 in the medium and high dose groups were significantly decreased （<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan can activate the PI3K/Akt/mTOR signaling pathway of granular cells， inhibit excessive autophagy of granular cells， improve ovarian function and insulin resistance， and restore ovulation， and the effect is better with high dose.
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OBJECTIVE：To study the effects of paeonol combined with cisplatin on the proliferation and apoptosis of human osteosarcoma cell MG- 63 and its possible mechanism. METHODS ：MG-63 cells in logarithmic growth phase were divided into blank control group ，cisplatin group （4 µmol/L），paeonol group （50 mg/L），and low ，medium，high concentration combined groups （50，100，200 mg/L paeonol+ 4 µmol/L cisplatin ）. CCK- 8 method was used to detect the cell proliferation rate at 24，48，and 72 hours of treatment. Annexin Ⅴ-FITC/PI double staining method was used to detect the cell apoptosis rate at 24 hours of treatment. The relative expression of PI 3KCA，Akt，mTOR，P-gp and PTEN mRNA in cells were detected by qRT-PCR. RESULTS ：Compared with blank control group ，the cell proliferation rate at each time point ，and the relative expression of PI 3KCA，Akt and mTOR mRNA in cells were significantly reduced and the apoptosis rate and the relative expression of P-gp and PTEN mRNA in the cells were increased significantly （P＜0.05 or P＜0.01）. Compared with cisplatin group and paeonol group ，cell proliferation rate at each time point and the relative expression of PI 3KCA，Akt and mTOR mRNA in cells were decreased significantly in the high concentration combination group ，while the relative expression of P-gp and PTEN mRNA in the cells were significantly increased （P＜0.01）；there were statistical significance in some of the above indicators in the medium and low concentration combination groups（P＜0.05 or P＜0.01）. CONCLUSIONS ：The combination of paeonol and cisplatin could inhibit the proliferation of MG- 63 cells and promote their apoptosis ，which may be related to the down-regulation of PI 3KCA，Akt and mTOR mRNA expression ，and the up-regulation of P-gp and PTEN mRNA expression.
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Objective:To evaluate the effect of resveratrol combined with γ-ray irradiation on the biological behavior of cervical cancer cells, and to explore its possible mechanism.Methods:The proliferation of cell populations after different concentrations of resveratrol solution±γ-ray irradiation was detected by CCK-8 assay. Scratch test and Transwell chamber test were used to detect cell migration and invasion. Flow cytometry and Annexin V-FITC/PI double staining were employed to assess cell apoptosis. Western blot was performed to measure the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR proteins.Results:Compared with the normal control (NC) group, the resveratrol group±γ-ray irradiation could inhibit the proliferation, migration, and invasion and promote cell apoptosis of human cervical cancer Hela cells, and the combined effect was more obvious. Compared with the NC group, resveratrol and γ-ray irradiation could significantly down-regulate the expression levels of Bcl-2, PI3K, p-Akt and p-mTOR proteins, up-regulate the expression level of Bax protein, but did not significantly alter the expression levels of Akt and mTOR proteins in human cervic1 255al cancer Hela cells.Conclusions:Resveratrol combined with γ-ray irradiation can dramatically inhibit the proliferation, migration, invasion, and promote the apoptosis of cervical cancer Hela cells. The mechanism may be related to the inhibition of the PI3K/Akt/mTOR signaling pathway and down-regulating the expression levels of downstream related proteins.
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@#[Abstract] Objective: To investigate the effect of cytokeratin 13 (CK13) on radio-sensitivity of human nasopharyngeal carcinoma HNE1 cell line and its mechanism. Methods: HNE1 cells were divided into control group, anti-CK13#a group (CK13 knockdown), anti-CK13#b group (CK13 knockdown), control+sirolimus group (100 nmol/L sirolimus treatment for 1 h), and anti-CK13#a + sirolimus group (100 nmol/L sirolimus treatment for 1 h). After irradiation treatment (200 cGy/min irradiation for 5 min), cell proliferation in each group was measured by CCK-8 assay. Cell apoptosis rate in each group was determined by Flow cytometry. Expression of PI3K/AKT/mTOR signaling pathway related PTEN gene was detected by qPCR, and WB was used to detect the expressions of PI3K/AKT/mTOR signaling pathway related proteins. Results: In the case of radiotherapy, as compared with the control group, the proliferation of HNE1 cells after CK13 knockdown was significantly enhanced (P<0.01) while the apoptosis rate was significantly reduced (P<0.01), the contents of caspase-3 and γH2AX as well as the protein lever of PTEN in cells were significantly decreased, while the expressions of p-AKT and p-S6K were significantly increased (all P<0.01). Interestingly, additional treatment with sirolimus (PI3K/AKT/mTOR signaling pathway inhibitor) could rescue the accelerated cell proliferation and decreased cell apoptosis caused by CK13 knockdown (all P<0.05). Conclusion: CK13 knockdown can enhance the activity of PI3K/AKT/mTOR signaling pathway by down-regulating PTEN, and ultimately reduce the radio-sensitivity of nasopharyngeal carcinoma HNE1 cells.
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@#[Abstract] Objective: To observe the effects of shikonin on the proliferation, apoptosis and cell cycle of human esophageal carcinoma TE-1 cells, and to explore its mechanism. Methods: TE-1 cells were treated with different concentrations of shikonin (0, 1, 5, 10 µmol/L). MTT assay was used to detect cell proliferation at different time points (24, 48 and 72 h). After treatment with shikonin for 48 h, cell apoptosis in TE-1 cells of each group was observed with Hoechst 33258 fluorescence staining. Flow cytometry was used to detect apoptosis and cell cycle. The changes in expression of TRAP1/Akt/mTOR signaling pathway related proteins were detected by Western blotting. Results: Shikonin inhibited the proliferation of TE-1 cells in a time-dose-dependent manner (P<0.05 or P<0.01). Compared with the control group, shikonin significantly promoted the apoptosis of TE-1 cells (P<0.01), induced the G0/G1 phase block of TE-1 cells (P<0.05 or P<0.01), and reduced the expression levels of TRAP1, p-Akt and p-MTOR (P<0.05 or P<0.01). The above effects were all dose-dependent. Conclusion: Shikonin can significantly inhibit the proliferation of TE-1 cells in vitro, induce G0/G1 phase arrest and promote apoptosis, which may be closely related to the inhibition of TRAP1/Akt/mTOR signaling pathway.
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OBJECTIVE@#To observe the effect of moxibustion on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin protein (PI3K/Akt/mTOR) signaling pathway in the foot-pad synovial tissue in rats with rheumatoid arthritis (RA), and to explore the mechanism of moxibustion for treating RA.@*METHODS@#Forty healthy SD rats were randomly divided into a control group, a model group, a moxibustion group, a cigarette-moxibustion group and a medication group, 8 rats in each group. The RA model was established with subcutaneous injection of complete Freund's adjuvant (CFA) in the left hind foot-pad under wind, cold and wet environment in the model group, the moxibustion group, the cigarette-moxibustion group and the medication group. The rats in the moxibustion group were treated with moxibustion at "Zusanli" (ST 36) for 20 min; the rats in the cigarette-moxibustion group were treated with moxibustion of ordinary cigarette at "Zusanli" (ST 36) for 20 min; the rats in the medication group were treated with tripterygium glycosides suspension (0.8 mg/100 g) by gavage. All the intervention was given once a day for 15 days. The left hind foot-pad volume was measured before and after modeling and after 15-day intervention. After 15-day intervention, the serum levels of IL-17 and IL-23 were detected by ELISA method, and the expression levels of PI3K, Akt and mTOR in synovial tissue of left hind foot-pad were detected by Western blot method.@*RESULTS@#The volume of left hind foot-pad, the serum levels of IL-23 and IL-17 and the expression of PI3K, Akt and mTOR in synovial tissue of left hind foot-pad in the model group were higher than those in the control group (@*CONCLUSION@#Moxibustion may play a therapeutic effect on RA by inhibiting the level of IL-23, IL-17 and the activity PI3K/Akt/mTOR, and regulating inflammatory response and autophagy.
Subject(s)
Animals , Rats , Arthritis, Rheumatoid/therapy , Moxibustion , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Rats, Sprague-Dawley , Signal Transduction , Synovial Membrane , TOR Serine-Threonine KinasesABSTRACT
Breast cancer (BC) is a commonly diagnosed cancer in females. MicroRNA-660-5p (miR-660-5p) has been reported to be involved in the occurrence and development of BC. However, the regulatory network of miR-660-5p in BC has not been fully addressed. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the enrichment of miR-660-5p and tet-eleven translocation 2 (TET2) in BC tissues and cells. Cell counting kit-8 (CCK8), flow cytometry, and transwell migration and invasion assays were used to measure cell proliferation, apoptosis, migration, and invasion. The target relationship between miR-660-5p and TET2 was confirmed by dual luciferase reporter assay. Protein expression was measured by western blot. The expression of miR-660-5p was elevated in BC, and high expression of miR-660-5p was closely related to lymph node metastasis, advanced TNM stage, and vascular invasion of BC tumors. miR-660-5p silencing inhibited cell proliferation and metastasis, but induced apoptosis of BC cells. TET2 was identified as a direct target of miR-660-5p, and the interference of TET2 partly reversed the suppressive effects of miR-660-5p silencing on the malignant potential of BC cells. miR-660-5p promoted BC progression partly through modulating TET2 and PI3K/AKT/mTOR signaling. miR-660-5p/TET2 axis might be a promising target for BC treatment.