Therapeutic potential of sulforaphane: modulation of NRF2 mediated PI3/AKT/mTOR pathway in oral fibrosis / Potencial terapêutico do sulforafano: modulação do NRF-2 mediado o pela via PI3/AKT/mTOR na fibrose oral

Oral Submucous Fibrosis is a potentially malignant disorder caused by habitual areca nut chewing, which contributes to the dispersion of active alkaloids into subepithelial tissues, stimulating excessive extracellular matrix deposition. Various treatment modalities are available; however, their efficacy in inhibiting fibrosis progression remains limited. Sulforaphane (SFN), an isothiocyanate found abundantly in cruciferous plants, is known to have effective antifibrotic properties. Objective: The present study investigated the antifibrotic effect of SFN via phosphatidylinositol 3 kinase (PI3K), Serine/Threonine Kinase 1 (AKT-1), mammalian target of rapamycin (mTOR) pathway in arecoline (AER) induced fibrosis in human gingival fibroblasts [HGFs]. Material and Methods: MTT assay determined the half-maximal inhibitory concentration of AER and SFN at 24h in the HGF cell line. Expression levels of transforming growth factor ß1 (TGFß1), collagen type 1 alpha 2 (COL1A2), hydroxyproline (HYP), PI3, AKT, mTOR, and nuclear factor erythroid 2­related factor 2 (NRF2) were assessed post-AER and SFN treatment using qPCR and western blot analysis. Results: The findings of the study revealed that AER elicited a stimulatory effect, upregulating TGFß1, COL1A2, HYP, PI3K, AKT, and mTOR and downregulating NRF2 expression. Conversely, SFN treatment significantly upregulated NRF2, inhibiting TGFß1 mediated PI3/AKT/mTOR pathway. Conclusion: These observations suggest that SFN can be used as a promising synergistic antifibrotic agent to combat fibrogenesis via the non-Smad pathway (AU)

Fibrose submucosa oral é uma desordem potencialmente maligna causada pelo habito de mascar a noz da areca, o que contribui para a dispersão de alcalóides ativos nos tecidos subepiteliais, estimulando a deposição excessiva de matriz extracelular. Há várias modalidades terapêuticas, no entanto, com eficácia limitada no controle da progressão da fibrose. O sulforafano (SFN), isotiocianato encontrado abundantemente em plantas crucíferas, é conhecido por suas propriedades antifibróticas. Objetivo: Investigar os efeitos antifibróticos do SFN na via fosfatidilinositol3-quinase (PI3K), via quinase serina/treonina 1 (AKT-1), via do alvo da rapamicina em mamíferos (mTOR), na fibrose induzida por arecolina (AER) em fibroblastos gengivais de humanos (HGFs). Material e Métodos: A meia concentração inibitória mínima de AER e SFN em 24 horas nas células HGFs foi determinada por MTT. Os níveis de expressão de ß1 (TGFß1), colágeno tipo 1 alfa 2 (COL1A2), hidroxiprolina (HYP), PI3K, AKT, mTOR, fator nuclear eritroide 2 relacionado ao fator 2 (NRF2) foram analisados após tratamento com ERA e SFN através de qPCR e western blot. Resultados: O ERA apresentou efeito estimulatório aumentando a expressão de TGFß1, COL1A2, HYP, PI3K, AKT e mTOR e diminuindo a expressão de NRF2. Por outro lado, tratamento com SFN aumentou significativamente a expressão de NRF2, inibindo a liberação de TGFß1 mediada pela via PI3/AKT/mTOR. Conclusão: Esses achados sugerem que o SFN pode ser um agente antifibrótico promissor no combate à fibrogênese decorrente da via não-Smad (AU)

Wenyang Jieyu Prescription Regulates Hippocampal Neuron Apoptosis and Improves Synaptic Plasticity in Depressed Mice via BDNF/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

Effects of osthole on renal oxidative stress injury in aged spontaneously hypertensive rats by regulating PI3K/Akt/mTOR signaling pathway / 中国临床药理学与治疗学

AIM: To explore the mechanism of osthole on elderly spontaneously hypertensive rats. METHODS: 20-month-old spontaneously hypertensive rats (SHRs) and healthy Wistar-Kyoto (WKY) rats were purchased. SHRs were treated with osthole (i.g.) for 8 weeks. The systolic blood pressure and diastolic blood pressure of rats were monitored. Hematoxylin-eosin staining (H&E), periodic acid-schiff staining (PAS) and Masson staining were used to observe the pathological changes of rat kidney tissues. The activity of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in rat kidney was detected by ELISA kit. PI3K/Akt/mTOR signaling pathway related proteins were detected by western blot. RESULTS: Osthole reduced the systolic and diastolic blood pressure of SHRs, improved the histopathological changes of SHRs kidney, reduced the activity of MDA in SHRs kidney, and increased the activity of SOD and GSH. Osthole reduced the levels of p-PI3K, p-Akt and p-mTOR. CONCLUSION: Osthole reduces the activity of PI3K/Akt/mTOR signaling pathway and exerts a protective effect on renal oxidative stress injury in aged spontaneously hypertensive rats.

Effect of Faeces Bombycis Extract on Cajal Interstitial Cells in Diabetic Gastroparesis Rats Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic mechanism of Faeces Bombycis on diabetic gastroparesis （DGP） rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein （PI3K/Akt/mTOR） signaling pathway. MethodDGP rat model was prepared by random selection of 15 out of 105 rats as blank group. The rats successfully constructed were randomly divided into model group， high-，medium- and low- dose groups （3.2， 1.6， 0.8 g·kg-1） and moxapride group （1.5 mg·kg-1）， with 12 rats in each group， and were given gavage for 4 weeks. The gastric emptying rate and random blood glucose were measured. The morphological changes of gastric antrum were observed by hematoxylin-eosin （HE） staining， and the expression of the c-Kit gene was analyzed by immunohistochemistry. The apoptosis of Cajal interstitial cells was observed by in situ end labeling （TUNEL） staining， and the protein expressions of PI3K， phosphorylation（p）-PI3K， Akt， p-Akt， mTOR， and p-mTOR were detected by Western blot. ResultCompared with the blank group， the gastric emptying rate of the model group decreased significantly （P<0.01）， and the glandular structure of the gastric antrum was destroyed. The expression of c-Kit decreased （P<0.01）， and the apoptosis of Cajal interstitial cells （ICC） increased. Compared with the model group， the gastric emptying rate in the high， middle， and low-dose groups of Faeces Bombycis extract and mosapride group increased significantly （P<0.01）. The glandular structure of the gastric antrum became closer， and the apoptosis of ICC decreased. The expression of c-Kit in the high dose group of Faeces Bombycis extract increased significantly. After Western blot testing， compared with the blank group， the protein expression of p-Akt/Akt， p-PI3K/PI3K， and p-mTOR/mTOR in the model group increased. Compared with the model group， the protein expression of p-Akt/Akt in the high dose group of Faeces Bombycis extract decreased （P<0.01）， and the protein expression of p-PI3K/PI3K decreased in the middle and low dose groups of Faeces Bombycis extract and mosapride group decreased （P<0.05， P<0.01）. The protein expression of p-mTOR/mTOR decreased in the low dose group of Faeces Bombycis extract （P<0.05）. In terms of random blood glucose， compared with the blank group， the random blood glucose in the model group increased significantly （P<0.01）， and compared with the model group， the random blood glucose in the high and middle dose groups of Faeces Bombycis extract decreased significantly （P<0.05）. Compared with mosapride group， the protein expression of p-Akt/Akt decreased in the high dose group of Faeces Bombycis extract （P<0.05）， and the protein expression of p-PI3K/PI3K increased in the high， middle， and low dose groups of Faeces Bombycis extract （P<0.05， P<0.01）. ConclusionFaeces Bombycis extract can increase gastric emptying rate， reduce ICC apoptosis， and lower random blood glucose in DGP rats. The high dose group of Faeces Bombycis extract has a significant effect on inhibiting ICC apoptosis， and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Magnolol and 5-fluorouracil synergy inhibition of metastasis of cervical cancer cells by targeting PI3K/AKT/mTOR and EMT pathways / 中草药·英文版

OBJECTIVE@#This study is designed to investigate the mode of action of the synergistic effect of 5-fluorouracil (5-FU) and magnolol against cervical cancer.@*METHODS@#Network pharmacological approach was applied to predict the molecular mechanism of 5-FU combined with magnolol against cervical cancer. CCK-8 assay, colony formation assay, immunofluorescence staining, adhesion assay, wound healing mobility assay, cell migration and invasion assay and Western blot analysis were conducted to validate the results of in silico study.@*RESULTS@#Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was identified as the key pathway in silico study. The experimental results showed that 5-FU combined with magnolol strongly inhibited cervical cancer cell proliferation, induced the morphological change of HeLa cells by down-regulating the expression of α-actinin, tensin-2 and vinculin. Moreover, magnolol enhanced inhibitory effect of 5-FU on the cell adhesion, migration and invasion. The phosphorylation of AKT and PI3K and the expression of mTOR were strongly inhibited by the combination of 5-FU and magnolol. Moreover, the expression of E-cadherin and β-catenin was upregulated and the expression of Snail, Slug and vimentin was down-regulated by the 5-FU together with magnolol.@*CONCLUSION@#Taken together, this study suggests that 5-FU combined with magnolol exerts a synergistic anti-cervical cancer effect by regulating the PI3K/AKT/mTOR and epithelial-mesenchymal transition (EMT) signaling pathways.

Association of PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms with pulmonary tuberculosis susceptibility in a Chinese population

ABSTRACT Background: Autophagy can inhibit the survival of intracellular microorganisms including Mycobacterium tuberculosis (Mtb), and the PI3K/AKT/mTOR pathway plays a crucial role. This study investigated the association between PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms and pulmonary tuberculosis (PTB) susceptibility. Methods: KEGG pathway and gene ontology (GO) databases were searched for genes belonging to the PI3K/AKT/mTOR and autophagy pathways. Thirty SNPs in nine genes were identified and tested for their associations with tuberculosis in 130 patients with PTB and 271 controls. We constructed genetic risk scores (GRSs) and divided the participants into 3 subgroups based on their GRSs:0-5, 6-10, and 11-16. Results: This analysis revealed that the AKT1 (rs12432802), RPTOR (rs11654508, rs12602885, rs2090204, rs2589144, and rs2672897), and TSC2 (rs2074969) polymorphisms were significantly associated with PTB risk. A decreasing trend was observed (P trend 0.020), in which a lower GRS was associated with a higher risk of PTB ([6-10] vs. [0-5]: OR (95%CI) 0.590 (0.374-0.931); [11-16] vs. [0-5]: OR (95%CI) 0.381 (0.160-0.906)). Conclusions: Polymorphisms in AKT1, RPTOR, and TSC2 may influence susceptibility to PTB.

S100A10 promotes proliferation and invasion of lung adenocarcinoma cells by activating the Akt-mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis.@*METHODS@#Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed.@*RESULTS@#The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis (P < 0.05), but not with tumor differentiation or the patients' age or gender (P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients (P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro (P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation (P < 0.001).@*CONCLUSION@#S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.

Methyl rosmarinate induces cell apoptosis in human hepatocellular carcinoma cells via inhibiting the Akt/mTOR signaling pathways / 西安交通大学学报(医学版)

【Objective】 To investigate the cell death-inducing effect of methyl rosmarinate (MR) on human hepatoma Hep-3B and SK-Hep1 cells and their potential mechanisms. 【Methods】 The effects of MR on the viability of Hep-3B, SK-Hep1 and MIHA cells were determined by cell counting kit-8 (CCK-8) assay. The morphological changes of three kinds of cells treated with different concentrations of MR were observed by optical microscopy. EdU assay and flow cytometry were used to detect the proliferation and apoptosis of Hep-3B and SK-Hep1 cells. Transwell assay was used to study the effects of MR on the migration and invasion of Hep-3B and SK-Hep1 cells. Western blotting was used to evaluate the protein expression levels of apoptosis, EMT and Akt/mTOR signaling pathways. 【Results】 After treated with different concentrations of MR (0~200 μmol/L) for 48 h, Hep-3B and SK-Hep1 cells activities were significantly decreased in a concentration-dependent manner (P<0.01), while there was no significant effect on MIHA cell activity (P>0.05), and the IC50 of Hep-3B and SK-Hep1 cells were 102.5 and 99.3 μmol/L, respectively. MR treatment (0-150 μmol/L) significantly inhibited the proliferation of Hep-3B and SK-Hep1 cells (P<0.05), while cell detachment and shrinkage were observed by optical microscopy on the Hep-3B and SK-Hep1 cells, while the morphology of MIHA cells was not changed. Compared with the control group, MR (100, 150 μmol/L) induced apoptosis in Hep-3B and SK-Hep1 cells, the expression levels of the pro-apoptotic proteins Bax and cleaved PARP were significantly increased (P<0.05), while the expression level of the anti-apoptotic protein Bcl-2 was significantly decreased (P<0.05). MR (100, 150 μmol/L) also inhibited the migration and invasion of HCC cells, significantly increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin compared with the control group (P<0.05). Finally, Western blotting results showed that the expressions of p-Akt and p-mTOR in Hep-3B and SK-Hep1 treated by MR were significantly reduced in a dose-dependent manner, suggesting that MR may play an anti-cancer role by inhibiting Akt/mTOR signaling pathway. 【Conclusion】 MR can promote apoptosis in Hep-3B and SK-Hep1 cells, which may be closely related to the inhibition of Akt/mTOR signaling pathway.

Mechanisms and research progress of targeted therapies on regulation of autophagy by natural compounds from traditional Chinese medicine / 药学学报

Autophagy is an important physiological process that can degrade cell components and maintain cell homeostasis, divided into three types including macroautophagy, microautophagy and chaperon-mediated autophagy generally, and macroautophagy is the most common form. Autophagy can affect the progression of a variety of diseases, such as cancer, neurodegenerative diseases, heart-related diseases, and autoimmune diseases, etc. However, autophagy can promote or inhibit diseases in different circumstances because of the dual roles of autophagy. Therefore, targeted regulating autophagy may be a potential treatment plan for diseases in specific stages of disease development. Now, with the development of traditional Chinese medicine (TCM) resources and the deepening of researches on the modern utilization of TCM, many active compounds from TCM have been discovered that can target autophagy to exert pharmacological activity. Most of the natural compounds activate or inhibit autophagy by affecting the classical PI3K/AKT/mTOR autophagy pathway. In addition, some compounds can also affect autophagy through MAPKs signaling pathways such as MEK/ERK, JNK and p38MAPK. These active compounds exert various biological activities by regulating autophagy, including anti-tumor, inhibiting neurodegenerative diseases, protecting cardiomyocytes, and relief of inflammatory response. In this review, we summarized the active compounds in TCM that affect autophagy by targeting different signaling pathways and their mechanisms of regulating autophagy, also introduced the effects of active compounds on diseases after affecting autophagy. Finally, this paper summarized and prospected the development of targeted autophagy for the treatment of diseases by TCM compounds, hoping to provide clues for subsequent exploration and research.

Effect of Clkl gene on level of Aβ-induced autophagy in Alzheimer's disease / 中国药理学通报

Aim To investigate the effect of siRNA transfection of silencing Clkl gene on autophagy levels in AD model cells. Methods The Clkl gene was silted using siRNA transfection techniques. MTT was used to observe the effects of Aβ

Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

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.

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.

Bushen Jianpi Prescription Regulates Autophagy in Patients with Aplastic Anemia / 中国实验方剂学杂志

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.

Wendantang Treats Inflammation in Obesity （Syndrome of Phlegm-dampness） by Regulating PI3K/Akt/mTOR Pathway-mediated Adipocyte Autophagy / 中国实验方剂学杂志

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.

Dihydromyricetin Alleviates H9C2 Cell Apoptosis and Autophagy by Regulating CircHIPK3 Expression and PI3K/AKT/mTOR Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effect and potential mechanism of dihydromyricetin (Dmy) on H9C2 cell proliferation, apoptosis, and autophagy.@*METHODS@#H9C2 cells were randomly divided into 7 groups, namely control, model, EV (empty pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector), IV (circHIPK3 interference), Dmy (50 µ mol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 II/I (LC3II/I), phospho-phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and phospho-mammalian target of rapamycin (p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells.@*RESULTS@#Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly (P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3 II/I significantly increased (all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3 II/I decreased significantly (all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced (P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed (P<0.05).@*CONCLUSION@#Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.

Eleutheroside B induces apoptosis and autophagy of lung cancer cells by regulating PI3K/Akt/mTOR pathway / 中国中药杂志

This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism. MTT assay was used to detect the cytotoxicity of eleutheroside B at 5, 10, 15, 20, 25, 30, 35, 40, and 45 mmol·L~(-1) on lung cancer cells. Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time. Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells. AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells, and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism. AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3. The results showed that compared with the control group, eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner. The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h, and the optimal concentrations were 28.64 and 22.16 mmol·L~(-1), respectively. Eleutheroside B could inhibit the colony formation of A549 and H460 cells. Compared with the control group, eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis, as well as induce the expression of mitochondrial pathway-related proteins. Under the effect of eleutheroside B, the acidic autophagy vacuole in lung cancer cells increased, LC3Ⅱ expression increased, P62 protein expression decreased, and PI3K, p-Akt, and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway. Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells, reduce colony formation, induce apoptosis of lung cancer cells through mitochondrial pathway, and induce autophagy. The mechanism may be related to the PI3K/Akt/mTOR pathway.

Protective effect of Shenfu Injection on regulation of autophagy in rats with chronic heart failure based on PI3K/Akt/mTOR pathway / 中国中药杂志

This study aimed to investigate the mechanism and target sites of Shenfu Injection in the intervention of chronic heart fai-lure based on the PI3K/Akt/mTOR autophagy signaling pathway. The chronic heart failure model was induced in rats by subcutaneous injection of isoproterenol. The model rats were randomly divided into model group, Shenfu Injection group, and 3-methyladenine autophagy inhibitor(3-MA) group. A normal group was also set up. After 15 days of administration, cardiac function indexes of the rats were detected by echocardiography. The serum N-terminal pro-B-type natriuretic peptide(NT-proBNP) levels were measured using the ELISA. HE and Masson staining was performed to observe the morphological changes in myocardial tissues, and electron microscopy was used to observe the autophagosomes in myocardial tissues. Western blot was conducted to measure the changes in autophagy-related proteins(LC3 Ⅱ/Ⅰ and p62), PI3K, Akt, mTOR, and phosphorylation levels. The results showed that compared with normal group, model group in rats led to reduced cardiac function, significant activation of cardiac autophagy, increased fibrotic lesions in myocardial tissues, structural disorder of the myocardium, increased autophagosomes, and cytoplasmic vacuolization. Compared with model group, Shenfu Injection group in rats led to cardiac function significantly improved, myocardial fibrosis decreased, and the number of autophagosomes and cytoplasmic vacuolization decreased. The phosphorylation levels of PI3K, Akt, and mTOR were significantly increased(P<0.01). In the 3-MA group, autophagy was inhibited through the activation of the PI3K/Akt/mTOR signaling pathway, resulting in improved cardiac function, reduced myocardial fibrosis, and no significant cytoplasmic vacuolization. The findings suggest that Shenfu Injection can activate the PI3K/Akt/mTOR signaling pathway and inhibit autophagy, thereby improving cardiac function.

Saikosaponin D regulates apoptosis and autophagy of pancreatic cancer Panc-1 cells via Akt/mTOR pathway / 中国中药杂志

This study aims to investigate the effect and mechanism of saikosaponin D on the proliferation, apoptosis, and autophagy of pancreatic cancer Panc-1 cells. The cell counting kit(CCK-8) was used to examine the effects of 7, 10, 13, 16, 19, 22, 25, and 28 μmol·L~(-1) saikosaponin D on the proliferation of Panc-1 cells. Three groups including the control(0 μmol·L~(-1)), low-concentration(10 μmol·L~(-1)) saikosaponin D, and high-concentration(16 μmol·L~(-1)) saikosaponin D groups were designed. The colony formation assay was employed to measure the effect of saikosaponin D on the colony formation rate of Panc-1 cells. The cells treated with saikosaponin D were stained with hematoxylin-eosin(HE), and the changes of cell morphology were observed. Hoechst 33258 fluorescent staining was used to detect the effect of saikosaponin D on the cell apoptosis. The autophagy staining assay kit with MDC was used to examine the effect of saikosaponin D on the autophagy of Panc-1 cells. Western blot and immunocytochemistry(ICC) were employed to examine the effect of saikosaponin D on the expression levels and distribution of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), cleaved caspase-3, autophagy-associated protein Beclin1, microtubule-associated protein light chain 3(LC3), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results showed that compared with the control group, saikosaponin D decreased the proliferation rate of Panc-1 cells in a dose-dependent and time-dependent manner. The colony formation rate of the cells significantly decreased after saikosaponin D treatment. Compared with the control group, the cells treated with saikosaponin D became small, accompanied by the formation of apoptotic bodies. The saikosaponin D groups showed increased apoptosis rate and autophagic vesicle accumulation. Compared with the control group, saikosaponin D up-regulated the expression of Bax, cleaved caspase3, Beclin1, LC3Ⅱ/LC3Ⅰ and down-regulated the expression of Bcl-2, caspase-3, p-Akt/Akt, and p-mTOR/mTOR. In addition, these proteins mainly existed in the cytoplasm. In conclusion, saikosaponin D can inhibit the proliferation and induce the apoptosis and autophagy of Panc-1 cells via inhibiting the Akt/mTOR pathway.

Mechanism of astragaloside Ⅳ in regulating autophagy of PC12 cells under oxygen-glucose deprivation by medicating Akt/mTOR/HIF-1α pathway / 中国中药杂志

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.

Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway / 中国中药杂志

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.