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.

Mechanism of Dihuang Yinzi in Improving Energy Metabolism Disorder and Autophagy Injury of Astrocytes in Brain of AD Mice / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Dihuang Yinzi （DHYZ）in improving astrocyte injury in the brain and regulating energy metabolism and autophagy disorder in Alzheimer's disease （AD） model mice. MethodForty male APP/PS1 transgenic mice aged four months were randomly divided into a model group and a model + DHYZ group （2.5 g·kg-1）， with 20 mice in each group. Forty C57BL/6J mice with the same background and same age were randomly divided into a control group and a control + DHYZ group （2.5 g·kg-1）， with 20 mice in each group. The mice in the control group and the model group were administered with an equal volume of sterilized normal saline by gavage， once a day for 150 days. Novel object recognition test and step-down test were performed to evaluate the learning and memory ability of mice. The expression of glial fibrillary acidic protein （GFAP） in astrocytes was detected by immunofluorescence and Western blot. High-performance liquid chromatography （HPLC） was used to detect adenosine triphosphate （ATP）， adenosine diphosphate （ADP）， and adenosine monophosphate （AMP） in brain tissues of mice， and the data obtained were used to calculate energy charge （EC） levels. The phosphorylation levels of liver kinase B1 （LKB1）， adenosine 5′-monophosphate （AMP）-activated protein kinase （AMPK）， UNC-51-like kinase 1 （ULK1）， and mammalian target of rapamycin （mTOR） and the expression levels of autophagy-related proteins Beclin-1， microtuble-associated protein 1 light chain 3 （LC3）-Ⅱ/LC3-Ⅰ， and p62 in mouse brain were measured by Western blot. ResultCompared with the control group， the model group showed decreased novel object recognition index， shortened retention latency， increased error times in the step-down test， up-regulated protein expression of GFAP， decreased content of ATP， ADP， and EC in brain tissues， elevated AMP ， increased levels of p-AMPK， p-LKB1， and p-mTOR， and protein expression of p62 ， and down-regulated p-ULK1 level and protein expression of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ（P<0.01）， while the above experimental indexes were not significantly different in the control + DHYZ group. Compared with the model group， the model + DHYZ group showed increased novel object recognition index（P<0.05）， prolonged retention latency（P<0.01）， decreased error times（P<0.01） in the step-down test， reduced protein expression of GFAP（P<0.05）， increased content of ATP， ADP， and EC in brain tissues （P<0.05， P<0.01）， decreased AMP content（P<0.05）， reduced p-AMPK， p-LKB1， and p-mTOR levels and protein expression of p62， and up-regulated p-ULK1 level and protein expression of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ（P<0.01）. ConclusionBy protecting astrocytes， DHYZ can improve energy metabolism and autophagy disorder in AD mice to improve the learning and memory ability of model mice.

The toxic effects of imidacloprid exposure on HepG2 cell based on non-targeted metabolomics / 环境与职业医学

Background Imidacloprid is a neonicotinoid insecticide that is widely used in agricultural production, with a high detection rate in human biological samples. Previous studies have shown a high correlation between imidacloprid exposure and liver injury, but the specific mechanism is still unknown. Objective To observe potential toxic effects of HepG2 cells and its perturbation of non-targeted metabolic profile after imidacloprid exposure, and to explore possible molecular mechanisms of hepatotoxicity of imidacloprid by analyzing invovlved biological processes and signaling pathways. Methods HepG2 cell suspension was prepared and seeded in a 96-well plate, which was divided into blank control group, dimethyl sulfoxide (DMSO) solvent control group and imidacloprid exposure groups with multiple concentrations. Each group was set with 5 parallel samples. The viability of HepG2 cells viability were determined after 8 h of exposure to different concentrationsof imidacloprid (1, 2.5, 5, 7.5, 10 mmol·L−1), and the dose-effect relationship was analyzed. A proper concentration (3 mmol·L−1 with 80% viability) was chosen for imidacloprid exposure, non-targeted metabolomic analysis was applied to the cultivated HepG2 cells using UHPLC-Q-TOF/MS technology, the differential metabolites between groups were screened, and the bioprocess and related signaling pathways of their enrichment were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results Compared to the other two groups, the survival rates of HepG2 cells in the imidacloprid exposure groups decreased. A survival rate of about 86% of HepG2 cells was found in HepG2 cells exposed to 2.5 mmol·L−1 imidacloprid exposure. The non-targeted metabolomics studies showed that 61 metabolites were significantly affected in HepG2 cells after 3 mmol·L−1 imidacloprid exposure, including creatine (variable importance in projection VIP=1.11, P<0.001), arginine (VIP=1.47, P=0.048), taurine (VIP=4.28, P=0.001), and α-D-glucose (VIP=1.90, P=0.006). The differential metabolites enriched in bioprocess and related signaling pathways were mainly directed to mTOR signaling pathways (P<0.001), arginine and proline metabolism (P=0.002), and galactose metabolism (P=0.015). Conclusion Imidacloprid exposure can significantly inhibit the survival rate of HepG2 cells, and interfere with the mTOR signaling pathway, arginine and proline metabolism, galactose metabolism, and so on.

Circulating miRNA-1-3p as Biomarker of Accelerated Sarcopenia in Patients Diagnosed with Chronic Heart Failure

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.

Comparison of effects of m-TOR inhibitors and calcineurin inhibitor on SUPAR and ox-LDL levels in renal transplant recipients / Comparación de los efectos de los inhibidores de m-TOR y el inhibidor de calcineurina sobre los niveles de SUPAR y ox-LDL en receptores de trasplante renal

ABSTRACT Introduction: We aimed to investigate the effect of different immunosuppressive regimens on SUPAR and ox-LDL levels which are early markers of inflammation in renal transplant recipients. Methods: A total number of 83 patients were enrolled in our study. While fourty- eight of those were received mTORi, thirty five patients were been receiving CNI. According to the immunosuppressive regimen patients were divided into CNI and m-TORi receving groups and serum SUPAR and ox-LDL levels were measured. Results: Log-SUPAR values were lower in the group receiving m-TORi (3.40 ± 0.1 vs 3.48 ± 0.4, p=0.010). OxLDL / LDL levels were higher (0.0168± 005 vs 0.0132 ±004, p=0.009) in the CNI group. In linear regression analysis, a statistically significant relationship was detected between the use of m-TORi and log-SUPAR (β = -0.052, 95% CI [-0.224, -0.012], p = 0.041) . A negative and independent relationship was found between HT and log-SUPAR (β = -0.60, 95% CI--0.112, -0.018], p=0.0024) and ox-LDL (β = -0.169 [-0.330, -0.008], p=0.040). Very strong correlation (r=1.0, p=<0.001) and independent relationship (β=0.321 [0.313,0.330], p=<0.001) was detected between ox-LDL and SUPAR. Conclusion: As a result, when compared immunsuppression between m-TORi and CNI, the former was associated with lower SUPAR and oxLDL levels.

RESUMEN Introducción: Nuestro objetivo fue investigar el efecto de diferentes regímenes inmunosupresores sobre los niveles de SUPAR y ox-LDL, que son marcadores tempranos de inflamación en receptores de trasplante renal. Material y métodos: Un total de 83 pacientes se inscribieron en nuestro estudio. Mientras que cuarenta y ocho de ellos recibieron mTORi, treinta y cinco pacientes recibieron CNI. De acuerdo con el régimen inmunosupresor, los pacientes se dividieron en grupos receptores de CNI y m-TORi y se midieron los niveles séricos de SUPAR y ox-LDL. Resultados: Los valores de Log-SUPAR fueron menores en el grupo que recibió m-TORi (3,40 ± 0,1 vs 3,48 ± 0,4, p = 0,010). Los niveles de OxLDL/LDL fueron mayores (0,0168± 005 vs 0,0132 ±004, p=0,009) en el grupo CNI. En el análisis de regresión lineal, se detectó una relación estadísticamente significativa entre el uso de m-TORi y log-SUPAR (β = -0,052, IC del 95% [-0,224, -0,012], p = 0,041). Se encontró una relación negativa e independiente entre HT y log-SUPAR (β = -0.60, 95% IC--0.112, -0.018], p = 0.0024) y ox-LDL (β = -0.169 [-0.330, -0.008], p = 0,040). Se detectó una correlación muy fuerte (r = 1,0, p <0,001) y una relación independiente (β = 0,321 [0,313, 0,330], p <0,001) entre ox-LDL y SUPAR. Conclusión: Como resultado, cuando se comparó la inmunosupresión entre m-TORi y CNI, la primera se asoció con niveles más bajos de SUPAR y oxLDL.

Down-regulation of autophagy proteins is associated with higher mTOR expression in the placenta of pregnant women with preeclampsia

Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining cellular integrity. It seems to be essential for cell survival during stress, starvation, hypoxia, and consequently to the placenta implantation and development. Preeclampsia (PE) is a multisystemic disorder characterized by the onset of hypertension associated or not with proteinuria and other maternal complications. Considering that the placenta seems to play an important role in the pathogenesis of PE, the objective of the present study was to evaluate protein levels of light chain protein (LC3), beclin-1, and the mammalian target of rapamycin (mTOR) in the placenta of pregnant women with PE. Placental tissues collected from 20 women with PE and 20 normotensive (NT) pregnant women were evaluated for LC3, beclin-1, and mTOR expression by qPCR and immunohistochemistry. The mRNA for LC3 and beclin-1 were significantly lower, while mTOR gene expression was significantly higher in the placenta of pregnant women with PE than in the NT group. Placentas of PE women showed significantly decreased protein expression of LC3-II and beclin-1, whereas mTOR was significantly increased compared with the NT pregnant women. There was a negative correlation between protein expression of mTOR and LC3-II in the placental tissue of PE women. In conclusion, the results showed autophagy deficiency suggesting that failure in this degradation process may contribute to the pathogenesis of PE; however, new studies involving cross-talk between autophagy and inflammatory molecular mechanisms might help to better understand the autophagy process in this obstetric pathology.

PI3K/Akt/mTOR signaling pathway in cutaneous squamous cell carcinoma / 中华皮肤科杂志

The pathogenesis of cutaneous squamous cell carcinoma (cSCC) is complicated, and treatment methods of advanced cSCC remain insufficient. This review summarizes the role of phosphatidylinositol 3-kinase (PI3K) /protein kinase B (Akt) /mammalian target of rapamycin (mTOR) signaling pathway in the pathogenesis of cSCC, as well as progress in the treatment of cSCC targeting this pathway, and provides new ideas for targeted therapy of cSCC.

Effect of Zuogui Jiangtang Tongmai Prescription on Astrocyte Injury by AGEs Combined with Oxygen-glucose Deprivation Based on AMPK/mTOR/ULK1 Pathway Related to Autophagy / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Zuogui Jiangtang Tongmai prescription （ZGJTTMP） on astrocytes （ASs） injured by advanced glycation end products（AGEs） combined with oxygen-glucose deprivation （OGD）. MethodCell counting kit-8 （CCK-8） was used to determine the optimal concentration of AGEs and the action time of OGD， and the optimal blood concentration of ZGJTTMP was selected for follow-up experiments. ASs were divided into normal group， model group （AGEs + OGD）， ZGJTTMP group， an adenosine 5'-monophosphate-activated protein kinase （AMPK） inhibitor （Compound C） group， AMPK activator （AICAR） group， and combination group （ZGJTTMP + AICAR）. The morphological changes in ASs in each group were observed under an inverted microscope. The cell survival rate in each group was detected by CCK-8. The content of interleukin-1β（IL-1β）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） was detected by enzyme-linked immunosorbent assay （ELISA）. The number of autophagosomes in each group was counted under an electron microscope. The expression of microtubule-associated protein light chain 3 （LC3） was observed by immunofluorescence. The protein expression of LC3， p62， p-AMPK， AMPK， p-mammalian target of rapamycin （mTOR）， mTOR， p-UNC-51 like kinase 1 （ULK1）， and ULK1 was detected by Western blot. ResultAccording to the results of cell survival rate， 200 mg·L-1 AGEs and OGD for 6 h were selected as the optimal modeling conditions for the model group， and 5% was selected as the optimal blood concentration of ZGJTTMP. Under the inverted microscope， the cells were severely damaged after modeling， but the cell injury in the ZGJTTMP group and the Compound C group was significantly improved. As revealed by ELISA results， the content of IL-1β， IL-6， and TNF-α in the model group increased （P<0.01）， and the content of inflammatory factors in the ZGJTTMP group and the Compound C group decreased （P<0.01）. Under the electron microscope， the number of autophagosomes in the model group increased significantly. The immunofluorescence results showed that the expression area of LC3 increased in the model group （P<0.01）， and the ZGJTTMP group and the Compound C group showed decreased number of autophagosomes and reduced expression area of LC3 （P<0.01）. As demonstrated by the results of Western blot， compared with the normal group， the model group showed increased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and decreased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. Compared with the model group， the ZGJTTMP group and the Compound C group showed decreased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and increased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. ConclusionZGJTTMP possesses a protective effect on ASs with inflammatory injury by AGEs combined with OGD， which may be achieved by inhibiting the activation of the AMPK/mTOR/ULK1 pathway related to autophagy， thus inhibiting the overexpression of autophagy.

Mechanism of Cucurbitacin B in Regulating Glycolysis and Inhibiting Proliferation of HuCCT1 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of cucurbitacin B （CuB） in inhibiting cell proliferation and glycolysis. MethodCell counting kit-8 （CCK-8） was applied to investigate the effect of different concentrations of CuB （0， 40， 80， 120， 160， 200， 400， and 800 nmol·L-1） on the proliferation of HuCCT1 cells. The effect of different concentrations of CuB （50， 100， and 200 nmol·L-1） on the colony formation ability of HuCCT1 cells was detected by plate cloning assay. The effect of different concentrations of CuB （50， 100， 200 nmol·L-1） on the HuCCT1 cell cycle was analyzed by flow cytometry. Visible spectrophotometry was employed to detect the activity of key glycolytic enzymes hexokinase （HK） and pyruvate kinase （PK）） and changes in glucose consumption， lactate production， and adenosine triphosphate （ATP） production in HuCCT1 cells after administration of different concentrations of CuB （50， 100， 200 nmol·L-1）. Western blotting was used to assay the effect of CuB on the expression of cell cycle-related proteins， proliferation-related proteins， key glycolytic proteins， and Akt/mammalian target of rapamycin （mTOR） pathway-related proteins. ResultAs compared with the blank group， CuB at dose of 160-800 nmol·L-1 after 24 h administration and CuB at dose of 80-800 nmol·L-1 after 48 h administration inhibited the proliferation of HuCCT1 cells in a time- and dose-dependent manner （P<0.05， P<0.01）， and the median inhibitory concentration was 200 nmol·L-1 48 h after administration. CuB can restrain the colony formation ability of HuCCT1 cells in a dose-dependent manner （P<0.01）， and block HuCCT1 cell cycle in G2 phase （P<0.05， P<0.01）. CuB （100 and 200 nmol·L-1） can suppress the activities of HK and PK and reduce cell glucose consumption and production of lactate and ATP （P<0.05， P<0.01）. Western blot results showed that CuB （100 and 200 nmol·L-1） can inhibit the protein levels of cycle-related protein Cyclin B1， proliferating cell nuclear antigen （PCNA）， HK1， HK2， PKM1， PKM2， phosphorylated Akt （p-Akt）， phosphorylated mTOR （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） （P<0.05， P<0.01）. ConclusionCuB can inhibit aerobic glycolysis in HuCCT1 cells via the Akt/mTOR pathway， thereby affecting cell proliferation.

Mechanism of Qihuang Yiqi Shexue Prescription in Treatment of ITP Model Mice Based on Autophagy Mediated by AMPK/mTOR/ULK1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Qihuang Yiqi Shexue prescription （QHYQSX） in the treatment of immune thrombocytopenia （ITP） model mice based on the autophagy mediated by the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase 1 （ULK1） signaling pathway. MethodFifty BALB/c mice were randomly divided into normal group， model group， high- and low-dose QHYQSX groups， and prednisone group， with 10 mice in each group. The ITP model was induced by intraperitoneal injection of anti-platelet serum （APS） of guinea pig. On the 8th day of the APS injection， drugs were administered by gavage for 14 days. Peripheral blood platelet （PLT） count and hemoglobin （Hb） concentration were detected. Spleen and thymus were separated， weighed， and the organ index was calculated. Sternum was sampled for bone marrow smear， and bone marrow megakaryocytes were classified under a microscope. Thrombopoietin （TPO）， interleukin-6 （IL-6）， IL-10， tumor necrosis factor-α （TNF-α）， transforming growth factor-β1 （TGF-β1）， and interferon-γ （IFN-γ） in the serum were detected by enzyme-linked immunosorbent assay（ELISA）. AMPK， mTOR， ULK1， microtubule-associated protein light chain 3 （LC3）， Beclin1， and p62 mRNA expression levels in the spleen were detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. The protein expression of AMPK， p-AMPK， p-mTOR， p-ULK1， LC3Ⅱ/LC3Ⅰ， Beclin1， and p62 in the spleen was detected by Western blot. ResultCompared with the normal group， the model group showed reduced peripheral blood PLT count， Hb， and TPO levels （P<0.05，P<0.01）， increased spleen and thymus indexes （P<0.01）， decreased number of bone marrow megakaryocytes （P<0.01）， elevated serum levels of IL-6， TNF-α， and IFN-γ （P<0.01）， and reduced IL-10 and TGF-β1 levels （P<0.01）. Compared with the model group， the groups with drug intervention showed increased PLT counts and TPO levels （P<0.01）， decreased spleen and thymus indexes （P<0.05， P<0.01）， elevated number of bone marrow megakaryocytes （P<0.05， P<0.01）， reduced serum levels of IL-6， TNF-α， and IFN-γ （P<0.05， P<0.01）， and up-regulated IL-10 and TGF-β1 levels （P<0.05，P<0.01）. Compared with the low-dose QHYQSX group， the high-dose QHYQSX group and the prednisone group showed different degrees of significant differences in improving PLT counts and levels of cellular inflammatory factors （P<0.05， P<0.01）. Real-time PCR and Western blot results showed that compared with the normal group， the model group showed up-regulated mRNA expression of AMPK， LC3， and Beclin1 and protein expression of p-AMPK/AMPK， LC3Ⅱ/LC3Ⅰ， and Beclin1 in the spleen （P<0.05， P<0.01）， and down-regulated mRNA expression of mTOR， ULK1， and p62 and protein expression of p-mTOR， p-ULK1， and p62 （P<0.05， P<0.01）. Compared with the results in the model group， high- and low-dose QHYQSX and prednisone could down-regulate the mRNA expression of AMPK， LC3， and Beclin1 and protein expression of p-AMPK/AMPK， LC3Ⅱ/LC3Ⅰ， and Beclin1 in the spleen （P<0.05， P<0.01）， and up-regulate the mRNA expression of mTOR， ULK1， and p62 and protein expression of p-mTOR， p-ULK1， and p62 （P<0.05， P<0.01）. ConclusionQHYQSX may inhibit excessive autophagy by regulating the AMPK/mTOR/ULK1 signaling pathway， thereby regulating immune intolerance and playing a role in the treatment of ITP.

Mechanism of Scutellariae Radix-Paeoniae Radix Rubra Combination of Different Proportions Against Hepatic Fibrosis in Rats / 中国实验方剂学杂志

ObjectiveTo observe the effects of Scutellariae Radix （SR）-Paeoniae Radix Rubra （PRR） combination of different proportions on the expression of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear transcription factor κB （NF-κB） and phosphatidylinositol kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathways in liver tissues of rats with hepatic fibrosis and explore the mechanism against hepatic fibrosis. MethodSixty male SD rats of SPF grade were randomly divided into a normal group， a model group， a positive control （silymarin） group， and SR-PRR 1∶1， SR-PRR 1∶2， and SR-PRR 1∶4 groups， with 10 rats in each group. The hepatic fibrosis model was induced in rats except for those in the normal group by intraperitoneal injection of 40% tetrachloromethane （CCl4）-olive oil solution at 3 mL·kg-1， 5 mL·kg-1 for the first time， for 8 weeks， twice per week. After 4 weeks， rats were treated correspondingly at 10 mL·kg-1 by intragastric administration， and the body weight of rats in each group was weighed for 8 weeks. After administration， histopathological changes in the liver were observed. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， hyaluronic acid （HA）， laminin （LN）， albumin （ALB）， alkaline phosphatase （AKP）， and superoxide dismutase （SOD） activities， malondialdehyde （MDA）， and hydroxyproline （HYP） content in liver tissues were detected. The mRNA expression levels of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， SR-PRR combination of different proportions could recover the body weight and improve the pathological injury of the liver. As revealed by enzyme linked immunosorbent assay （ELISA） results， compared with the normal group， the model group showed increased ALT， AST， HA， LN， AKP， MDA， and HYP levels to different degrees （P<0.05）. Compared with the model group， the groups with drug intervention showed decreased levels of ALT， AST， HA， LN， AKP， MDA， and HYP， potentiated SOD activity， and increased level of ALB （P<0.05）. As revealed by Real-time PCR results， compared with the normal group， the model group showed increased mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR （P<0.05）. Compared with the model group， the groups with drug intervention showed reduced mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats （P<0.05）. ConclusionSR-PRR combination of different proportions can improve the histopathological injury in liver tissues caused by CCl4， with the optimal effect observed in the SR-PRR 1∶4 group. SR-PRR may inhibit the development of liver fibrosis by inhibiting the expression of TLR4/MyD88/NF-κB and PI3K/Akt/mTOR signaling pathways， thereby alleviating chemical-induced liver injury.

Neurorotective Effect of Chaihu Jia Longgu Mulitang on Parkinson's Disease with Depression Model Rats and Its Mechanism Based on AMPK/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the protective effect of Chaihu Jia Longgu Mulitang （CLMT） on dopaminergic neurons in Parkinson's disease with depression （PDD） model rats， and to explore the mechanism based on adenosine monophosphate-activated protein kinase/mammalian target of rapamycin （AMPK/mTOR） signaling pathway. MethodAmong the 80 male SD rats， 10 were randomly selected as normal group and the rest were treated with long-term low-dose subcutaneous injection of rotenone in the neck and back combined with chronic unpredictable mild stress （CUMS） to establish PDD rat model. The successfully modeled PDD rats were randomly divided into model group， western medicine group （madopar 0.032 g·kg-1+fluoxetine hydrochloride 0.002 g·kg-1）， CLMT low-dose， medium-dose and high-dose groups （5， 10 and 20 g·kg-1）， 10 rats in each group. Normal group and model group were administrated with the same amount of normal saline by gavage for 4 consecutive weeks. Behavioral changes of rats in each group were evaluated by open field test and pole climbing test. The content of dopamine （DA） and 5-hydroxytryptamine （5-HT） in cerebrospinal fluid was determined by high performance liquid chromatography （HPCL）. The pathological changes of dopaminergic neurons in substantia nigra of rats were observed by hematoxylin-eosin （HE） staining. The positive expression of tyrosine hydroxylase （TH） and expression of α-synuclein in substantia nigra were detected by immunohistochemistry （IHC） and immunofluorescence （IF）， repsectively. The protein expression of microtubule-associated protein 1 light chain 3 （LC3）， adenosine monophosphate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， mammalian target of rapamycin （mTOR） and phosphorylated mTOR （p-mTOR） was detected by Western blot. ResultCompared with the conditions in normal group， the total horizontal distance and the activity time in the central region in open field test and the content of DA and 5-HT in cerebrospinal fluid were decreased （P<0.05， P<0.01）， and the time of pole climbing was shortened （P<0.01）， with increased score （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group increased the total horizontal distance and activity time in the central region and the content of DA and 5-HT （P<0.05， P<0.01）， and extended the time of climbing pole （P<0.05）， with decreased score （P<0.05， P<0.01）. Compared with those in normal group， the number of dopaminergic neurons in the substantia nigra was reduced， with narrowed and loosely arranged cell body. The fluorescence expression of α-synuclein was enhanced （P<0.01）， and the positive expression of TH was decreased （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group showed elevated number of dopaminergic neurons in the substantia nigra， with enlarged cell body， and decreased fluorescence expression of α-synuclein， and enhanced the positive expression of TH （P<0.05， P<0.01）. Compared with normal group， model group had lowered expression of LC3Ⅱ/Ⅰ， p-AMPK/AMPK in striatum （P<0.05， P<0.01） and increased expression of p-mTOR/mTOR （P<0.01）. Compared with those in model group， LC3Ⅱ/Ⅰ and p-AMPK/AMPK expression were increased （P<0.05， P<0.01） and p-mTOR /mTOR expression was decreased （P<0.01） in CLMT high-dose group and western medicine group. ConclusionCLMT exerts a neuroprotective effect by inhibiting rotenone neurotoxicity. It enhances the level of DA， and thus improves the depression condition in rats with Parkinson's disease. The underlying mechanism may be related to the regulation of AMPK/mTOR signaling pathway， activation of autophagy， and promotion of degrading α-synuclein.

Protection of Zuoguiwan Against Apoptosis of Follicles in Rats Injured by 60Co-γ Rays： Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

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.

Protection of Zuoguiwan Against Apoptosis of Follicles in Rats Injured by 60Co-γ Rays： Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

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.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Research advances on the role of mTOR signaling pathway in regulating autophagy in spinal cord injury / 中国医师杂志

Traumatic spinal cord injury (SCI) has devastating effects on patients′ physical and mental health. Autophagy is widely involved in various physiological and pathological processes of the body and plays a key role in spinal cord injury. The mammalian target of rapamycin (mTOR) is the main regulator of autophagy. mTOR regulation of autophagy is closely related to the pathological process of spinal cord injury, and can effectively promote the recovery of spinal cord injury. Therefore, mTOR is a promising target for treatment of spinal cord injury. This article reviews the role of mTOR signal transduction pathway in the regulation of autophagy in spinal cord injury, in order to provide reference for follow-up research.

Study on the mechanism of mTOR/HIF-1α signaling pathway in Budd-Chiari syndrome liver fibrosis / 国际外科学杂志

Objective:To explore the mechanism of mTOR/HIF-1α signaling pathway in Budd-Chiari syndrome (B-CS) liver fibrosis.Methods:Twenty male C57 mice were randomly divided into Sham operation group (Sham), sham operation+ rapamycin (Sham+ Ra) group, B-CS group, B-CS+ rapamycin (B-CS+ Ra) Group, 5 in each group. The B-CS mouse model was constructed by partial ligation of the inferior vena cava(IVC) at the posterior segment of the liver; IVC was not ligated in the Sham group. Mice in Sham+ Ra and B-CS+ Ra groups were intraperitoneally injected with rapamycin (2 mg/kg, 5% DMSO solution preparation) every other day, Sham group and B-CS group were injected with the same dose of 5% DMSO solution.After 6 weeks, samples were taken, and part of the liver tissue was used to make paraffin sections for hematoxylin-eosin (HE) and Sirus Red staining to observe the pathological changes, and immunohistochemical staining to detect the expression of α-SMA and Fibrinogen in liver tissues; Protein and RNA were extracted from fresh liver tissue, and Western-blot was used to detect α-SMA, Fibrinogen, p-mTOR, mTOR, HIF-1α, Collagen I, and VEGF protein levels. Real-time fluorescent quantitative PCR was used to detect mTOR, HIF-1α, CollagenⅠ, VEGF mRNA levels.Measurement data were expressed as mean±standard deviation ( ± s), and the comparison between groups was performed by one-way ANOVA test. Results:The results of pathological staining showed that in the B-CS group, there was severe congestion around the central vein of the liver and sinusoids, widening of the sinus space, and increased collagen deposition, indicating that this study successfully established a mouse B-CS liver fibrosis model. The expression levels of fibrosis indicators α-SMA and Collagen I protein, mTOR pathway related indicators p-mTOR and HIF-1α protein, and microthrombus indicator Fibrinogen protein in the Sham group were 0.027±0.012, 0.337±0.008, 0.138±0.024, 0.296±0.113, 0.733±0.192; B-CS group were 0.986±0.001, 0.927±0.055, 0.936±0.044, 1.693±0.443, 1.612±0.068, and the differences were statistically significant ( P<0.05). The expression levels of B-CS+ Ra group were 0.707±0.078, 0.311±0.024, 0.332±0.094, 0.254±0.117, 0.569±0.075, which were statistically significant compared with B-CS group ( P<0.05). Conclusions:The mTOR/HIF-1α signaling pathway is significantly activated in mouse B-CS liver fibrosis. This pathway may participate in the development of liver fibrosis by regulating microthrombosis.

Intravenous route to choroidal neovascularization by macrophage-disguised nanocarriers for mTOR modulation

Retinal pigment epithelial (RPE) is primarily impaired in age-related macular degeneration (AMD), leading to progressive loss of photoreceptors and sometimes choroidal neovascularization (CNV). mTOR has been proposed as a promising therapeutic target, while the usage of its specific inhibitor, rapamycin, was greatly limited. To mediate the mTOR pathway in the retina by a noninvasive approach, we developed novel biomimetic nanocomplexes where rapamycin-loaded nanoparticles were coated with cell membrane derived from macrophages (termed as MRaNPs). Taking advantage of the macrophage-inherited property, intravenous injection of MRaNPs exhibited significantly enhanced accumulation in the CNV lesions, thereby increasing the local concentration of rapamycin. Consequently, MRaNPs effectively downregulated the mTOR pathway and attenuate angiogenesis in the eye. Particularly, MRaNPs also efficiently activated autophagy in the RPE, which was acknowledged to rescue RPE in response to deleterious stimuli. Overall, we design and prepare macrophage-disguised rapamycin nanocarriers and demonstrate the therapeutic advantages of employing biomimetic cell membrane materials for treatment of AMD.

A novel PI3K inhibitor XH30 suppresses orthotopic glioblastoma and brain metastasis in mice models

Glioblastoma is carcinogenesis of glial cells in central nervous system and has the highest incidence among primary brain tumors. Brain metastasis, such as breast cancer and lung cancer, also leads to high mortality. The available medicines are limited due to blood-brain barrier. Abnormal activation of phosphatidylinositol 3-kinases (PI3K) signaling pathway is prevalent in glioblastoma and metastatic tumors. Here, we characterized a 2-amino-4-methylquinazoline derivative XH30 as a potent PI3K inhibitor with excellent anti-tumor activity against human glioblastoma. XH30 significantly repressed the proliferation of various brain cancer cells and decreased the phosphorylation of key proteins of PI3K signaling pathway, induced cell cycle arrest in G1 phase as well. Additionally, XH30 inhibited the migration of glioma cells and blocked the activation of PI3K pathway by interleukin-17A (IL-17A), which increased the migration of U87MG. Oral administration of XH30 significantly suppressed the tumor growth in both subcutaneous and orthotopic tumor models. XH30 also repressed tumor growth in brain metastasis models of lung cancers. Moreover, XH30 reduced IL-17A and its receptor IL-17RA in vivo. These results indicate that XH30 might be a potential therapeutic drug candidate for glioblastoma migration and brain metastasis.

EGCG and ECG induce apoptosis and decrease autophagy via the AMPK/mTOR and PI3K/AKT/mTOR pathway in human melanoma cells / 中国天然药物

Catechins have been proven to exert antitumor effects in different kinds of cancers. However, the underlying mechanisms have not been completely clarified yet. This study aimed to assess the effects and mechanisms of (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) on human melanoma skin A375 cells. Results showed that EGCG and ECG inhibited the proliferation of A375 cells and ECG showed better inhibitory effect. Flow cytometry analysis had shown that EGCG and ECG induced apoptosis and led to cell cycle arrest. EGCG and ECG decreased Bcl-2 expression and upregulated Caspase-3 protein level, indicating the development of apoptosis. Furthermore, EGCG and ECG could decreased mitochondrial membrane potential of A375 cells. In addition, the expression of Beclin-1, LC3 and Sirt3 were downregulated at protein levels, which known to be associated with autophagy. After autophagy was increased by rapamycin, the apoptotic trend was not change, indicating that apoptosis and autophagy are independent. Mechanistically, EGCG and ECG treatments decreased phosphorylated-AMPK (p-AMPK) and increased the ratios of p-PI3K, p-AKT and p-mTOR in melanoma cells. Conclusively, EGCG and ECG induced apoptosis via mitochondrial signaling pathway, downregulated autophagy through modulating the AMPK/mTOR and PI3K/AKT/mTOR signaling pathway. It indicated that EGCG and ECG may be utilized in human melanoma treatment.