Betulinic Acid Induces Autophagy and Apoptosis of Human Colorectal Cancer SW620 Cells by Regulating PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of betulinic acid （BA） on apoptosis and autophagy of human colorectal cancer SW620 cells and the regulatory role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodCell viability was detected by methyl thiazolyl tetrazolium （MTT） colorimetry to determine the optimal administration time and dosage for subsequent experiments. Four groups were designed， including blank group and low-， medium-， and high-dose BA groups. Hematoxylin-eosin （HE） staining was conducted for the observation of SW620 cell morphology， and annexin-V/propidium iodide double staining for the determination of apoptosis rate in SW620 cells. Hoechst33258 staining and MDC staining were used for the observation of apoptosis and autophagy， respectively. Western blotting was employed to determine the protein levels of B-cell lymphoma/leukemia-2（Bcl-2）-associated X protein （Bax）， aspartate proteolytic enzyme-9 （Caspase-9）， activated aspartate proteolytic enzyme-3 （cleaved Caspase-3）， microtubule-associated protein 1 light chain 3 （LC3）， the mammalian homolog of yeast Atg6 （Beclin-1）， p62， phosphorylated PI3K （p-PI3K）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） in SW620 cells. ResultBA inhibited the activity of SW620， HT29， and HCT116 cells in a concentration- and time-dependent manner. The cells treated with BA for 48 h had lower viability than those treated for 24 h （P<0.05， P<0.01）. The half maximal inhibitory concentration (IC50） value of BA at the time point of 48 h was also lower than that at the time point of 24 h （P<0.01）， and that for SW620 cells was the minimum. BA induced the apoptosis in a concentration-dependent manner and increased the autophagosomes. Compared with the blank group， BA increased the apoptosis rate （P<0.01）， up-regulated the protein levels of Bax， Caspase-9， cleaved Caspase-3， and LC3 Ⅱ （P<0.05， P<0.01）， and down-regulated the protein levels of p62， p-Akt， p-PI3K， and p-mTOR （P<0.01）. Additionally， medium- and high-dose BA up-regulated the protein level of beclin-1 （P<0.01）. ConclusionBA may inhibit the activity of SW620 cells by hindering the PI3K/Akt/mTOR signaling pathway to induce cell apoptosis and autophagy.

Effect of Fuzitang on Proliferation of Human Rheumatoid Arthritis Synovial Fibroblast Cell Line MH7A and Expression of miR-155 / 中国实验方剂学杂志

ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.

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.

Effect of Huangqisan Pellets on PI3K/Akt/mTOR Signaling Pathway and Autophagy in Kidney of Diabetic Nephropathy Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Huangqisan pellets （HQS） on the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway and autophagy in the kidney of diabetic nephropathy （DN） rats. MethodDN rat model was established through high-fat diet combined with intraperitoneal injection of streptozotocin （35 mg·kg-1）. DN rats were randomly assigned into model group， irbesartan （0.027 g·kg-1） group， low-dose HQS （0.54 g·kg-1） group and high-dose HQS （1.08 g·kg-1） group. The levels of 24 h urinary total protein （UTP）， serum albumin （Alb）， serum creatinine （SCr）， urea nitrogen （BUN）， triglyceride （TG） and total cholesterol （TC） were measured after 12 weeks of continuous administration. The pathological changes of renal tissue were observed via hematoxylin-eosin （HE） staining. The expression of podocyte split diaphragm proteins nephrin and podocin in the renal tissue were detected by immunohistochemistry. The protein levels and phosphorylation of key proteins in PI3K/Akt/mTOR signaling pathway， as well as the expression of yeast Atg6 homolog （Beclin1） and microtubule-associated protein 1 light chain 3 （LC3） in the renal tissue were analyzed by Western blot. ResultCompared with the control group， the model group showcased increased 24 h UTP， SCr， BUN， TG， and TC levels and decreased Alb level （P<0.01）. After modeling， the rats showed granulosity of epithelial cells of renal tubules， thickening of capillary basement membrane， proliferation of mesangial cells， and sclerosis of glomerulus. Furthermore， modeling down-regulated the expression of nephrin and podocin in the podocyte hiatus of glomerulus （P<0.01） as well as the protein levels of p-PI3K， p-Akt， and p-mTOR and the autophagy markers LC3 and Beclin1 in renal tissue （P<0.01）. Compared with model group， irbesartan and HQS decreased the 24 h UTP， Cr， BUN， TG， and TC levels， increased the Alb level， and alleviated the pathological damage of kidney. Moreover， they up-regulated the expression of Nephrin and Podocin in the podocyte hiatus of glomerulus， as well as the protein levels of p-PI3K， p-Akt， p-mTOR， LC3， and Beclin1 in renal tissue （P<0.05， P<0.01）. ConclusionHQS may inhibit the PI3K/Akt/mTOR signaling pathway to enhance podocyte autophagy and protect the glomerulus， thus slowing down the development of DN.

Intervention Mechanism of Xiaoluowan on Experimental Goiter Rats Based on PI3K/Akt/mTORC1 Pathway / 中国实验方剂学杂志

ObjectiveBy observing the effect of Xiaoluowan on phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin complex 1 （mTORC1） pathway in experimental goiter rats， this study aims to explore its therapeutic effect on experimental goiter rats. MethodSixty 5-month-old SD rats of SPF grade were purchased， half males and half females， of which 10 were used as a normal group， and the remaining rats were administrated with propylthiouracil （PTU） solution to induce nodular goiter. After successful modeling， rats were randomly divided into a model group， levothyroxine sodium tablets group， Xiaoluowan low-dose group， medium-dose group， and high-dose group， ten rats each. The levothyroxine sodium tablets group was given 15 μg·kg-1 levothyroxine sodium tablets by gavage. The Xiaoluowan low-， medium-， and high-dose groups were given （ig） Xiaoluowan low-dose （10 g·kg-1）， medium-dose （20 g·kg-1）， and high-dose （30 g·kg-1） Xiaoluowan， and the normal group and model group were administered （ig） with the same volume of 0.9% sodium chloride solution. Four weeks after the intervention， rats were sacrificed by routine intraperitoneal anesthesia using 5% phenobarbital. Subsequently， the histopathology was observed under a microscope， and serum thyroid hormone levels were measured using a Roche electrochemiluminescence immunoassay analyzer. Serum cytokines were detected by enzyme-linked immunosorbent assay （ELISA）， and neurotransmitters were measured using a high-performance liquid chromatograph. The protein level of PI3K/Akt/mTORC1 pathway was determined by Western blot. ResultAs compared with the normal group， the levels of basic fibroblast growth factor （bFGF）， vascular endothelial growth factor （VEGF）， insulin-like growth factor （IGF-1）， 5-hydroxytryptamine （5-HT）， and thyroid stimulating hormone （TSH） were increased， and PI3K， Akt， and mTORC1 protein levels were up-regulated in the model group， while the levels of norepinephrine （NE）， triiodothyronine （T3）， tetraiodothyronine （T4）， free triiodothyronine （FT3）， and free thyroid hormone （FT4） were decreased （P<0.05）. Compared with the model group， the levothyroxine sodium tablets group， and Xiaoluowan low-， medium-， and high-dose groups exhibited reduced levels of bFGF， VEGF， IGF-1， 5-HT， and TSH， and down-regulated PI3K， Akt， and mTORC1 protein levels， and increased NE， T3， T4， FT3， and FT4 levels （P<0.05）. ConclusionXiaoluowan may act on the PI3K/Akt/mTORC1 signaling pathway to play its role in the treatment of nodular goiter， and it is dose-dependent.

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.

Bile acid coordinates microbiota homeostasis and systemic immunometabolism in cardiometabolic diseases

Cardiometabolic disease (CMD), characterized with metabolic disorder triggered cardiovascular events, is a leading cause of death and disability. Metabolic disorders trigger chronic low-grade inflammation, and actually, a new concept of metaflammation has been proposed to define the state of metabolism connected with immunological adaptations. Amongst the continuously increased list of systemic metabolites in regulation of immune system, bile acids (BAs) represent a distinct class of metabolites implicated in the whole process of CMD development because of its multifaceted roles in shaping systemic immunometabolism. BAs can directly modulate the immune system by either boosting or inhibiting inflammatory responses via diverse mechanisms. Moreover, BAs are key determinants in maintaining the dynamic communication between the host and microbiota. Importantly, BAs via targeting Farnesoid X receptor (FXR) and diverse other nuclear receptors play key roles in regulating metabolic homeostasis of lipids, glucose, and amino acids. Moreover, BAs axis per se is susceptible to inflammatory and metabolic intervention, and thereby BAs axis may constitute a reciprocal regulatory loop in metaflammation. We thus propose that BAs axis represents a core coordinator in integrating systemic immunometabolism implicated in the process of CMD. We provide an updated summary and an intensive discussion about how BAs shape both the innate and adaptive immune system, and how BAs axis function as a core coordinator in integrating metabolic disorder to chronic inflammation in conditions of CMD.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB-P300-BRD4 axis

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)-P300-bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid-liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

The cGAS-STING signaling in cardiovascular and metabolic diseases: Future novel target option for pharmacotherapy

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling exert essential regulatory function in microbial-and onco-immunology through the induction of cytokines, primarily type I interferons. Recently, the aberrant and deranged signaling of the cGAS-STING axis is closely implicated in multiple sterile inflammatory diseases, including heart failure, myocardial infarction, cardiac hypertrophy, nonalcoholic fatty liver diseases, aortic aneurysm and dissection, obesity, etc. This is because of the massive loads of damage-associated molecular patterns (mitochondrial DNA, DNA in extracellular vesicles) liberated from recurrent injury to metabolic cellular organelles and tissues, which are sensed by the pathway. Also, the cGAS-STING pathway crosstalk with essential intracellular homeostasis processes like apoptosis, autophagy, and regulate cellular metabolism. Targeting derailed STING signaling has become necessary for chronic inflammatory diseases. Meanwhile, excessive type I interferons signaling impact on cardiovascular and metabolic health remain entirely elusive. In this review, we summarize the intimate connection between the cGAS-STING pathway and cardiovascular and metabolic disorders. We also discuss some potential small molecule inhibitors for the pathway. This review provides insight to stimulate interest in and support future research into understanding this signaling axis in cardiovascular and metabolic tissues and diseases.

Therapeutic regulation of autophagy in hepatic metabolism

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ that governs body energy metabolism. Autophagy's role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.

Electroacupuncture in the treatment of neurogenic urine retention through autophagy mediated by AMPK/mTOR pathway / 中南大学学报(医学版)

OBJECTIVES@#Electroacupuncture can enhance autophagic flow, promote neuronal regeneration, axonal and myelin remodeling to achieve the protection of spinal cord injury, but its role in neurogenic urine retention is not completely clear. This study aims to investigate whether the mechanism of electroacupuncture in the treatment of neurogenic urine retention is through autophagy mediated by adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway.@*METHODS@#A rat model of neurogenic urine retention after sacral spinal cord injury was established. The rats with successful model were randomly divided into a model group, an electroacupuncture group (electro-acupuncture for Ciliao, Zhongji, and Sanyinjiao by electronic stimulation, once a day, 20 min each time for 7 days), and an electroacupuncture+AMP-activated protein kinase (AMPK) inhibitor group (on the basis of the treatment of electroacupuncture group, 100 μg of AMPK inhibitor compound C was injected intramuscularly around the L2-3 intervertebral space on the 1st and 4th day). The normal group did not receive any treatment. The maximum bladder volume, bladder basal pressure, leak point pressure, and bladder compliance were recorded by multi-channel physiological recorder; the morphology of bladder tissue was observed by HE staining; autophagy was observed under transmission electron microscope; the expressions of LC3II and Beclin1 protein were observed by immunofluorescence staining; the protein levels of AMPK, phosphorylated-AMPK (p-AMPK), mTOR, phosphorylated-mTOR (p-mTOR), microtubule associated protein 1 light chain 3 (LC3) II and Beclin1 in bladder tissue were detected by Western blotting.@*RESULTS@#Compared with the normal group, the maximum bladder capacity, leak point pressure, bladder compliance, p-AMPK, LC3II, Beclin1 protein expressions in the bladder tissue of the model group increased, and the p-mTOR protein expressions were decreased (all P<0.05); compared with the model group, the maximum bladder capacity, bladder compliance, p-mTOR protein expression in the bladder tissue of the electroacupuncture group were decreased, and the p-AMPK, LC3II, and Beclin1 protein expressions were increased (all P<0.05); compared with the electroacupuncture group, the maximum bladder capacity, bladder compliance, p-mTOR protein expression in the bladder tissue of the electroacupuncture+AMPK inhibitor group were increased, the p-AMPK, LC3II, and Beclin1 protein expressions were decreased (all P<0.05). In the model group, the bladder became larger, with unclear and varying degrees of degeneration, severe tissue damage and autophagosome appeared; the bladder of the electroacupuncture group was smaller than that of the model group, and all levels were clearly visible with autophagy bodies; the layers were slightly disordered and damaged in the electroacupuncture + AMPK inhibitor group.@*CONCLUSIONS@#Electroacupuncture can activate autophagy through AMPK/mTOR pathway, thereby reducing neurogenic urine retention caused by spinal cord injury.

Research progress on AMPK/mTOR signaling pathway involved in immunometabolic regulation in sepsis / 中国小儿急救医学

The occurrence, development and prognosis of sepsis are closely related to immune regulation.Immunometabolism has been the research hotspot of immune intervention in sepsis in recent years.AMP-activated protein kinase(AMPK)and mammalian target of rapamycin(mTOR)are star molecules involved in metabolic regulation.As an important way of immunometabolic regulation in sepsis, AMPK-mTOR is involved in the process of chemotaxis of neutrophils, the polarization of macrophages, the development and differentiation of natural killer cells and dendritic cells, and the development and functional regulation of T cells.This article reviewed the research progress of AMPK-mTOR signaling pathway on regulating metabolic reprogramming in immune cells, which contributes to immunoregulation in sepsis.

Effects of overexpression of LncRNA MEG3 on autophagy, apoptosis and mTOR pathway in pancreatic cancer cells PANC1 / 中华内分泌外科杂志

Objective:To investigate the effects of overexpression of long non-coding RNA maternally expressed gene 3 (LncRNA MEG3) on autophagy, apoptosis and mammalian rapamycin target protein (mTOR) pathway in pancreatic cancer cells (PANC1) .Methods:The pCMV-N-Flag-MEG3 expression plasmid was constructed and transfected into PANC1 cells. The expression of LncRNA MEG3 in hpde6c7 (normal pancreatic cells) group, PANC1 (blank control) , Vector (PANC1 cell transfected empty vector) group and MEG3 (PANC1 cell transfected with pCMV-N-Flag-MEG3 recombinant plasmid) group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) ; methyl thiazolyl tetrazolium (MTT) , flow cytometry and monodansylcadaverin (MDC) staining were used to detect the effects of overexpression of LncRNA MEG3 on the proliferation, apoptosis and autophagy of PANC1 cells; Western blot was used to detect the effects of overexpression of LncRNA MEG3 on the expression levels of Bcl-2, Bax and Beclin-1 in PANC1 cells, and the phosphorylation levels of mTOR, ribosomal p70S6 kinase protein (SK61) and uclear initiation factor 4E binding protein 1 (4E-BP1) in mTOR pathway.Results:Compared with those in PANC1 group and Vector group, the expression level of LncRNA MEG3 (0.36±0.08 vs 0.35±0.11 vs 0.69±0.09) , proliferation inhibition rate (3.35%±0.12 vs 3.23%±0.09 vs 36.77%±0.13) , autophagy rate (29.32%±1.03 vs 26.73%±1.32 vs 57.76%±1.09) , apoptosis rate (9.85%±1.58 vs 9.73%±1.12 vs 35.89%±1.05) , expression levels of Bax (0.26±0.08 vs 0.29±0.05 vs 0.83±0.08) and Beclin 1 (0.15±0.06 vs 0.17±0.02 vs 0.61±0.03) of PANC1 cells in MEG3 group were significantly higher (all P<0.05) , and the expression level of Bcl-2 (0.79±0.12 vs 0.81±0.09 vs 0.30±0.03) and phosphorylation levels of mTOR (1.08±0.05 and 1.06±0.08 vs 0.37±0.10) , SK61 (1.12±0.06 and 1.11±0.09 vs 0.41±0.03) and 4E-BP1 (0.97±0.07 and 0.95±0.03 vs 0.39±0.05) in mTOR pathway were significantly lower (all P<0.05) . Conclusion:Overexpression of LncRNA MEG3 can inhibit the proliferation of PANC1 cells, promote apoptosis and formation of autophagic vesicles, which may be related to the blocking of mTOR pathway.