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

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

Research progress in risk factors of post-transplantation diabetes mellitus / 器官移植

Solid organ transplantation has significantly prolonged the survival of patients with end-stage diseases. However, long-term use of immunosuppressants will increase the risk of post-transplantation diabetes mellitus (PTDM) in the recipients, thereby elevating the risk of infection, cardiovascular disease and death. In recent years, with persistent improvement of diagnostic criteria of PTDM, clinicians have deepened the understanding of this disease. Compared with type 2 diabetes mellitus, PTDM significantly differs in pathophysiological characteristics and clinical progression. Hence, different treatment strategies should be adopted. Early identification of risk factors of organ transplant recipients, early diagnosis and intervention are of significance for improving the quality of life of recipients, prolonging the survival of grafts and reducing the fatality of recipients. Therefore, the diagnosis, incidence and risk factors of PTDM were reviewed in this article, aiming to provide reference for clinicians to deliver prompt diagnosis and intervention for PTDM.

Protective Mechanism of Paeoniflorin on Mice with Ulcerative Colitis Based on AMPK/mTOR Autophagy Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg-1）， paeoniflorin （50 mg·kg-1） + inhibitor （20 mg·kg-1） group， and high dose （50 mg·kg-1）， medium dose （25 mg·kg-1）， and low dose （12.5 mg·kg-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Comparison of Effect of Hirudo， Notoginseng Radix et Rhizoma， and Their Combinations on Renal Fibrosis in Rats with Chronic Renal Failure / 中国实验方剂学杂志

ObjectiveTo observe the effects of Hirudo， Notoginseng Radix et Rhizoma， and drug pair on renal pathological morphology and protein phosphatase 2A （PP2A）/adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signal pathway in rats with chronic renal failure （CRF）. MethodThe 55 male SD rats were randomly divided into a normal group （n=11） and a modeling group （n=44）. The normal group was fed conventionally， and the modeling group was given 0.25 g·kg-1·d-1 adenine by gavage for 28 days to replicate the CRF model. After successful modeling， rats were randomly divided into model group， Hirudo group （3 g·kg-1·d-1）， Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， and Hirudo + Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， with 9 rats in each group. The normal group and model group were given a constant volume of normal saline by intragastric administration for 30 days. At the end of the experiment， the levels of serum creatinine （SCr） and urea nitrogen （BUN） in all groups were measured. The renal pathological morphology changes were observed by hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy. The mRNA expressions of PP2A， AMPK， and mTOR were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of PP2A， AMPK， phosphorylation（p）-AMPK， mTOR， and p-mTOR in renal tissue were detected by Western blot. ResultCompared with the normal group， the renal pathological structure changes were obvious， and the levels of SCr and BUN were significantly increased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression were significantly increased， and the p-AMPK/AMPK was significantly decreased in the model group （P<0.05）. Compared with the model group， the renal pathological morphology changes were significantly improved， and the levels of SCr and BUN were significantly decreased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression in the renal tissue were significantly decreased， and the p-AMPK/AMPK was significantly increased （P<0.05） in all groups after drug intervention. In addition， the effect in the Hirudo+Notoginseng Radix et Rhizoma group was better. The mRNA expression levels of AMPK and mTOR in the renal tissue were not significantly different among the normal group， model group， and other groups. ConclusionThe efficacy of Hirudo and Notoginseng Radix et Rhizoma pairs in improving renal fibrosis in rats with CRF is significantly better than that of the single drug， and its improvement on renal fibrosis in rats with CRF may be related to the regulation of PP2A/AMPK/mTOR signaling pathway.

Danggui Shaoyaosan Regulates Autophagy via AMPK/mTOR/ULK1 Signaling Pathway in Rat Model of Metabolism-associated Fatty Liver Disease / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase-1 （ULK1） signaling pathway in the rat model of metabolism-associated fatty liver disease （MAFLD）. MethodSixty SD rats were randomized into control， model， western medicine （polyene phosphatidylcholine capsules，0.144 g·kg-1）， and low-， medium-， and high-dose （2.44， 4.88， 9.76 g·kg-1， respectively） Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks， the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment， serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group， the model group showed increased contents of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C） and activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum， increased contents of TC， TG， and free fatty acids （FFAs） in the liver （P<0.01）， and decreased content of high-density lipoprotein cholesterol （HDL-C） in the serum （P<0.01）. Furthermore， the model group showed down-regulated protein levels of p-AMPK， microtubule-associated protein 1 light chain 3B （LC3B） Ⅱ， Beclin1， and ULK1 （P<0.01） and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver （P<0.01）. The hepatic steatosis was obvious and the NAFLD activity score （NAS） and oil red O staining area increased in the model group， （P<0.05， P<0.01）. Compared with the model group， Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum， lowered the levels of TC， TG， and FFA in the liver， down-regulated the protein levels of p-mTOR and p62 （P<0.01）， elevated the serum HDL-C level， and up-regulated the protein levels of p-AMPK， LCBⅡ， Beclin1， and ULK1 in the liver （P<0.05， P<0.01）. Moreover， it alleviated hepatic steatosis and decreased the NAS and oil red O staining area （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.

Biological principles for "homotherapy for heteropathy" / 药学学报

Non-infectious chronic diseases in human including diabetes, non-alcoholic fatty liver disease (NAFLD), atherosclerosis (AS), neurodegenerative diseases, osteoporosis, as well as malignant tumors may have some common pathogenic mechanisms such as non-resolved inflammation (NRI), gut microbiota dysfunction, endoplasmic reticulum stress, mitochondria dysfunction, and abnormality of the mammalian target of rapamycin (mTOR) pathway. These pathogenic mechanisms could be the basis for "homotherapy for heteropathy" in clinic. Some commonly used clinical drugs, such as metformin, berberine, aspirin, statins, and rapamycin may execute therapeutic effect on their targeted diseases,and also have the effect of "homotherapy for heteropathy". The mechanisms of the above drugs may include anti-inflammation, modulation of gut microbiota, suppression of endoplasmic reticulum stress, improvement of mitochondria function, and inhibition of mTOR. For virus infectious diseases, as some viruses need certain commonly used replicases, the inhibitors of the replicases become examples of "homotherapy for heteropathy" for antiviral therapy in clinic (for example tenofovir for both AIDS and HBV infection). Especially, in case of outbreak of new emerging viruses, these viral enzyme inhibitors such as azvudine and sofibuvir, could be rapidly used in controlling viral epidemic or pandemic, based on the principle of "homotherapy for heteropathy". In this review article, we show the research progress of the biological basis for "homotherapy for heteropathy" and the possible mechanisms of some well-known drugs, in order to provide insights and new references for innovative drug R&D.

Mechanism of Dahuangtang pellets in regulating podocyte autophagy of diabetic nephropathy mice through AMPK/mTOR/ULK1 signaling pathway / 中国临床药理学与治疗学

AIM: To explore the intervention effect of Dahuangtang pellets (DHT) on diabetic nephropathy (DN) based on the AMP-activated protein kinase/mammalian target of rapamycin/unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. METHODS: Eight mice were randomly assigned to the model group, the dapagliflozin group, and the DHT (high, medium, and low dosage) group out of a total of 40 C57BL/KSJ-db/db (hereafter referred to as db/db) mice; another 10 C57BL/KSJ-db/dm mice were used as the normal group, saline was provided to the normal and model groups, and the mice in the treatment group received the appropriate medications. The medications were given for 10 consecutive weeks, once per day, to the mice in the treatment group. At weeks 0, 4, 8, and 10 of administration, fasting blood glucose (FBG) was assessed by drawing blood at a predetermined time from the tail vein; Urine samples were taken at 0, 5, and 10 weeks after treatment to evaluate the levels of albumin and creatinine, and the urinary albumin-creatinine ratio (ACR) was computed. After 10 weeks, mice in each group were assayed for 24 h total urine protein, serum creatinine (Scr), urea nitrogen (BUN) levels; Western blotting analysis was conducted to detect the expression of p-AMPK, p-mTOR, and p-ULK1, as well as the expression of autophagy related proteins homolog of yeast Atg6 (Beclin-1), autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3), P62 in renal tissue; Immunohistochemistry was used to measure the expression of podocyte lacunar membrane proteins (Nephrin, Podocin) in renal tissues; The pathological morphology of renal tissue was observed by light microscopy and transmission electron microscopy. RESULTS: Compared with the model group, FBG, ACR, and 24 h total urine protein were reduced in the dapagliflozin group and DHT groups of mice, and there was no statistically significant difference in Scr and BUN; In renal tissues, there is increased expression of p-AMPK and p-ULK1, decreased expression of p-mTOR, increased expression of LC3II / LC3I and Beclin-1, and decreased expression of P62 (P<0.01, P< 0.05); differentially upregulated in glomeruli are the podocyte lacunar membrane proteins Nephrin and Podocin (P<0.01, P<0.05); renal pathologic damage was reduced to varying degrees; transmission electron microscopy showed an increase in the number of autophagic vesicles and autophagic lysosomes. CONCLUSION: DHT can delay the development of DN by regulating the AMPK / mTOR / ULK1 signaling pathway, enhancing podocyte autophagy, and protecting glomeruli.

Mechanism of Shenshuai Xiezhuo Decoction in Intervention of Chronic Kidney Disease Combined with Atherosclerosis Based on Regulation of PI3K/Akt/mTOR Signaling Pathway by miRNA126 / 中国实验方剂学杂志

ObjectiveExploring the role of microRNA126 （miRNA126） in chronic kidney disease combined with atherosclerosis （CKD AS） by regulating the phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway and the mechanism of Shenshuai Xiezhuo decoction in the intervention of CKD AS rats with 5/6 nephrectomy combined with high-fat feeding. MethodA total of 60 SD rats were randomly divided into sham operation group， model group， losartan group， and low， medium， and high dose groups of Shenshuai Xiezhuo decoction. The CKD AS rat model was established by 5/6 nephrectomy combined with high-fat feeding for 10 weeks. The low， medium， and high dose groups （6.0， 12.0， 24.0 g·kg-1·d-1） of Shenshuai Xiezhuo decoction and the losartan group （20 mg·kg-1·d-1） were gavaged， and the corresponding intervention was carried out for eight weeks. Then， the rats were killed， and samples were collected for corresponding detection. Fully automated biochemical analyzers were used to detect kidney function and blood lipids in rats： blood creatinine （SCr）， blood urea nitrogen （BUN）， total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） levels. Hematoxylin-eosin （HE） and Masson staining of aortic tissue and pathological observation under a light microscope were carried out， and autophagosomes and autophagy lysosomes were observed by transmission electron microscopy. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to determine the mRNA levels of miRNA126， PI3K， Akt， and mTOR in rats， and Western blot was used to determine the protein expression levels of phosphorylated （p）-PI3K， PI3K， p-Akt， Akt， p -mTOR， mTOR， benzyl chloride 1 （Beclin-1）， and microtubule-associated protein light chain 3Ⅱ/Ⅰ （LC3Ⅱ/LC3Ⅰ）. ResultCompared with the sham operation group， the serum SCr， BUN， TC， TG， and LDL-C in the model group were significantly increased （P<0.01）. Compared with the model group， the SCr， BUN， TC， TG， and LDL-C were decreased in the losartan group and low， medium， and high dose groups of Shenshuai Xiezhuo decoction （P<0.05）. Compared with the sham operation group， thickening plaques， infiltration of mononuclear macrophages， a small number of foam cells， disordered arrangement of smooth muscle fibers in the tunica media， and increased collagen fibers were observed in the model group， and the lesions in the losartan group and Shenshuai Xiezhuo decoction groups were alleviated compared with those in the model group. Compared with the model group， the number of autophagosomes and autophagy lysosomes increased in the medium and high dose groups of Shenshuai Xiezhuo decoction. Compared with the sham operation group， the expression of miRNA126 in the aortic tissue of the model group was significantly decreased （P<0.01）， and the mRNA expressions of PI3K， Akt， and mTOR were significantly increased （P<0.01）. Compared with the model group， the expression of miRNA126 in the aortic tissue of rats in high， medium， and low dose groups of Shenshuai Xiezhuo decoction and losartan group was significantly increased （P<0.01）， while the mRNA expressions of PI3K， Akt， and mTOR were significantly decreased （P<0.01）. Compared with the sham operation group， the protein expressions of p-PI3K， PI3K， p-Akt， Akt， p-mTOR， and mTOR in the model group were significantly increased （P<0.01）， while the protein levels of Beclin-1， LC3Ⅰ， and LC3Ⅱ were significantly decreased （P<0.01）. Compared with the model group， the protein expressions of p-PI3K， PI3K， p-Akt， Akt， p-mTOR， and mTOR in the losartan group and low， medium， and high dose groups of Shenshuai Xiezhuo decoction were decreased （P<0.05）， while the protein levels of Beclin-1 and LC3Ⅱ/LC3Ⅰ were increased （P<0.05）. ConclusionThe expression of miRNA126 is decreased in the aortic tissue of CKD AS rats， and the PI3K/Akt/mTOR pathway is activated to inhibit autophagy flux. Shenshuai Xiezhuo decoction regulates the PI3K/Akt/mTOR signaling pathway through miRNA126， restores the autophagy of aortic endothelial cells， protects the damage of CKD vessels， reduces the formation of As plaques， and slows the development of cardiovascular complications.

Effect of Qihuang Jianpi Zishen Granules on STAT3/mTOR Signaling Pathway in Patients with Systemic Lupus Erythematosus / 中国实验方剂学杂志

ObjectiveTo investigate the clinical efficacy of Qihuang Jianpi Zishen Granules in the treatment of systemic lupus erythematosus （SLE） and its effect on the signal transducer and activator of tranSCription 3/mammalian target of rapamycin （STAT3/mTOR） signaling pathway， and to decipher the possible mechanism. MethodSixty female SLE patients who met the criteria in the First Affiliated Hospital of Anhui University of Chinese Medicine from May 2022 to May 2023 were selected and randomized into a control group and an observation group （30 cases in each group）. The control group was treated with prednisone acetate + hydroxychloroquine sulfate orally， and the observation group was additionally treated with Qihuang Jianpi Zishen granules. The treatment lasted for 8 weeks. The SLE disease activity （SLEDAI）， TCM syndrome score， erythrocyte sedimentation rate （ESR）， hypersensitive C-reactive protein （hs-CRP）， immune indexes ［immunoglobulin G （IgG）， C3， C4， CD4+， and CD8+］， interleukin （IL）-17， IL-23， interferon （IFN）-γ， 24 h urinary protein （24 h PRO）， serum creatinine （SCr）， and expression of proteins ［STAT3， phosphorylated （p）-STAT3， mTOR protein and STAT3，mTOR mRNA］ in the STAT3/mTOR signaling pathway were determined before and after treatment. In addition， the adverse reactions were recorded. ResultAfter 8 weeks of treatment， the total response rate in the observation group was 93.33% （28/30）， which was higher than that （70.00%， 21/30） in the control group （χ2=4.007， P<0.05）. After treatment， both groups showed declined SLEDAI， TCM syndrome score， ESR， hs-CRP， IgG， CD8+， IL-17， IL-23， IFN-γ， 24 h PRO， SCr， and expression of proteins in the STAT3/mTOR pathway （P<0.01） and elevated levels of C3， C4， and CD4+ （P<0.01）. Moreover， the observation group had lower SLEDAI， TCM syndrome score， ESR， hs-CRP， IgG， CD8+， IL-17， IL-23， IFN-γ， 24 h PRO， SCr， and expression of proteins in the STAT3/mTOR pathway （P<0.05， P<0.01） and higher levels of C3， C4， and CD4+ （P<0.05， P<0.01） than the control group after treatment. Neither group showed serious adverse reactions during the treatment period. ConclusionQihuang Jianpi Zishen Granules can ameliorate the inflammatory response， reduce the disease activity， and mitigate the kidney injury in SLE by inhibiting the STAT3/mTOR signaling pathway to regulate the immune function.

Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway.@*METHODS@#Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR.@*RESULTS@#The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01).@*CONCLUSIONS@#EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

Dichloroacetic acid and rapamycin synergistically inhibit tumor progression / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD+‍) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells' response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells' susceptibility to rapamycin treatment in vitro and in vivo.

Baicalin Ameliorates Corticosterone-Induced Depression by Promoting Neurodevelopment of Hippocampal via mTOR/GSK3β Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the role of hippocampal neurodevelopment in the antidepressant effect of baicalin.@*METHODS@#Forty male Institute of Cancer Research mice were divided into control, corticosterone (CORT, 40 mg/kg), CORT+baicalin-L (25 mg/kg), CORT+baicalin-H (50 mg/kg), and CORT+fluoxetine (10 mg/kg) groups according to a random number table. An animal model of depression was established by chronic CORT exposure. Behavioral tests were used to assess the reliability of depression model and the antidepressant effect of baicalin. In addition, Nissl staining and immunofluorescence were used to evaluate the effect of baicalin on hippocampal neurodevelopment in mice. The protein and mRNA expression levels of neurodevelopment-related factors were detected by Western blot analysis and real-time polymerase chain reaction, respectively.@*RESULTS@#Baicalin significantly ameliorated the depressive-like behavior of mice resulting from CORT exposure and promoted the development of dentate gyrus in hippocampus, thereby reversing the depressive-like pathological changes in hippocampal neurons caused by CORT neurotoxicity. Moreover, baicalin significantly decreased the protein and mRNA expression levels of glycogen synthase kinase 3β (GSK3β), and upregulated the expression levels of cell cycle protein D1, p-mammalian target of rapamycin (mTOR), doublecortin, and brain-derived neurotrophic factor (all P<0.01). There were no significant differences between baicalin and fluoxetine groups (P>0.05).@*CONCLUSION@#Baicalin can promote the development of hippocampal neurons via mTOR/GSK3β signaling pathway, thus protect mice against CORT-induced neurotoxicity and play an antidepressant role.

Astragaloside Ⅳ Induces Autophagy and Apoptosis in Nasopharyngeal Carcinoma Cells via PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and underlying molecular mechanism of astragaloside-Ⅳ （AS-Ⅳ） on autophagy and apoptosis of nasopharyngeal carcinoma cells. MethodIn experiments in vitro， the effect of AS-Ⅳ on the autophagy of nasopharyngeal carcinoma cells was observed by monodansylcadaverine （MDC） staining and transmission electron microscopy （TEM）. In experiments in vivo， immunofluorescence （IF） and Western blot were used to detect the changes in autophagy and apoptosis and the expression of key proteins in the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway after the establishment of a xenograft tumor model in nude mice. ResultAfter 5-8F cells were treated with AS-Ⅳ of different doses （5， 10， 20 μmol·L-1）， the fluorescence intensity of autophagy in AS-Ⅳ groups significantly increased as compared with that in the blank group. The fluorescence expression of autophagy in AS-Ⅳ groups was the strongest after intervention for 24 hours， and the fluorescence expression in the 10 μmol·L-1 AS-Ⅳ group was the most obvious. The autophagy activator rapamycin （RAPA） induced more autophagosomes in 5-8F cells under the transmission electron microscope， and 3-methyladenine （3-MA）， an autophagy inhibitor， did not induce autophagosome formation in 5-8F cells under the transmission electron microscope as compared with the results in the blank group. In the 10 μmol·L-1 AS-Ⅳ group， the intracellular structure and cell membrane were intact and clear， and autophagosome formation was observed. Compared with the blank group， the AS-Ⅳ groups showed inhibited tumor volume （P<0.05， P<0.01）， potentiated fluorescence signals of microtubule-associated protein l light chain 3 type Ⅱ/microtubule-associated protein l light chain 3 type Ⅰ （LC3 Ⅱ/Ⅰ） and cleaved Caspase-3 （P<0.05， P<0.01）， increased expression levels of the mammalian homolog of yeast ATG6 （Beclin-1）， LC3 Ⅱ/Ⅰ， cleaved Caspase-3， and cleaved PARP （P<0.05， P<0.01）， down-regulated expression of ubiquitin-binding protein （p62） （P<0.05， P<0.01）， and reduced protein expression levels of phosphorylated PI3K （p-PI3K）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） （P<0.05， P<0.01）. ConclusionAS-Ⅳ can induce autophagy and apoptosis of nasopharyngeal carcinoma cells， and the mechanism is presumably attributed to the activation of the PI3K/Akt/mTOR signaling pathway.

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

ObjectiveTo explore the effects of Bushen Jianpi prescription on the autophagy and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway in the patients with aplastic anemia （AA）. MethodA total of 30 AA patients admitted to Xiyuan Hospital and 6 healthy donors who were prepared to undergo peripheral blood hematopoietic stem cell transplantation in 304 Hospital from September 2020 to August 2021 were enrolled and assigned into an AA group and a control group. The AA group was treated with Bushen Jianpi prescription combined with cyclosporin A （CsA） and androgen for 3 months. The mononuclear cells from bone marrow in the AA group before and after treatment and the peripheral blood of the control group were collected. Transmission electron microscopy was then employed to detect autophagosomes. Western blotting was employed to determine the protein levels of microtuble-associated protein 1 light chain 3 （LC3）Ⅰ， LC3Ⅱ， mTOR， phosphorylated （p）-mTOR， Akt， p-Akt， PI3K， and p-PI3K， and real-time polymerase chain reaction （PCR） to determine the mRNA levels of LC3， mTOR， Akt， and PI3K. ResultIn the AA group， the treatment was completed in 29 patients， and the total response rate was 51.72% （15/29）. ① The AA group showed lower levels of white blood cell （WBC）， hemoglobin （HGB）， platelet （PLT）， and absolute neutrophil count （ANC） in the peripheral blood （P<0.01） and lower number of intracellular autophagosomes than the control group before treatment. Moreover， the AA group showed lower mRNA level of LC3 （P<0.01） and protein levels of LC3Ⅰ and LC3Ⅱ （P<0.01） and higher mRNA levels of mTOR， Akt， and PI3Kα （P<0.01） and protein levels of Akt， p-Akt， PI3K， p-PI3K， mTOR， and p-mTOR （P<0.01） than the control group. ② In AA group， the levels of HGB and PLT elevated （P<0.05） and the number of intracellular autophagosomes increased after treatment compared with those before treatment. Moreover， the mRNA level of LC3 and the protein levels of LC3Ⅰ and LC3Ⅱ were up-regulated （P<0.01）， the mRNA levels of mTOR， Akt， and PI3Kα （P<0.01） and the protein levels of Akt， p-PI3K （P<0.01）， p-Akt， PI3K， mTOR， p-mTOR （P<0.05） were down-regulated after treatment. ConclusionAA patients show lower autophagy levels， while Bushen Jianpi prescription can effectively improve the autophagy level and down-regulated the expression of PI3K/Akt/mTOR signaling pathway in AA patients.

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

ObjectiveTo observe the effects of Wendantang on the expression of inflammatory cytokines， autophagy markers， and key molecules of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway in the adipocytes of the rat model of obesity （syndrome of phlegm-dampness） and to explore the material basis of inflammation in obesity （syndrome of phlegm-dampness） and the underlying mechanism of Wendantang intervention. MethodA total of 126 SD rats were randomized into 2 groups： 16 rats in the blank group and 110 rats in the modeling group. The blank group was fed with a basic diet while the modeling group with a high-fat diet to establish the animal model of obesity （syndrome of phlegm-dampness） for 8 weeks. After successful modeling， 48 obese rats were selected according to their body mass and randomized into a model control group， an orlistat （ORLI， 32.40 mg·kg-1） group， a rapamycin （RAPA， 2 mg·kg-1） group， and low-， medium-， and high-dose （4.45， 8.90， 17.80 g·kg-1， respectively） Wendantang groups， with 8 rats in each group. In addition， 8 rats were randomly selected from the blank group to be set as the normal control group. The corresponding agents in each group were administrated by gavage and the model and control groups were administrated with equal amounts of distilled water once daily for 6 weeks. The body mass， Lee's index， body fat ratio， and obesity rate were measured or calculated. The expression of UNC51-like kinase-1 （ULK1）， Beclin1， human autophagy-related protein 5 （Atg5）， p62， and microtubule-associated protein 1 light chain 3 （LC3） Ⅰ/Ⅱ （markers of autophagy in adipocytes） was detected by the immunohistochemical two-step method. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the expression of tumor necrosis factor （TNF）-α， interleukin-6 （IL-6）， IL-1β， monocyte chemotactic protein-1 （MCP-1）， IL-4， IL-10， IL-13， and transforming growth factor （TGF）-β in adipocytes. Western blot was employed to measure the protein levels of classⅠ-PI3K， phosphatidylinositol triphosphate （PIP3）， Akt， mTORC1， ULK1， TSC1， and TSC2 in adipocytes. ResultCompared with the blank group， the modeling group showed increased body mass and Lee's index （P<0.01）， the obesity rate >20%， and phlegm-dampness syndrome manifestations such as physical obesity， decreased mobility， decreased appetite， lusterless and tight fur， loose stools， decreased responsiveness to the outside world， and decreased water intake. Compared with the normal control group， the model control group showed increased body mass， Lee's index， body fat ratio， adipocyte autophagy marker expression， pro- and anti-inflammatory cytokine levels （P<0.05， P<0.01）， down-regulated protein levels of classⅠ-PI3K， PIP3， Akt， mTORC1， TSC1， and TSC2 （P<0.01）， and up-regulated protein level of ULK1 （P<0.01）. The intervention groups showed lower body mass， body fat ratio， adipocyte autophagy marker protein expression， and protein levels of TNF-α， IL-6， IL-1β， MCP-1， IL-4， and IL-13 than the model control group （P<0.05， P<0.01）. Moreover， the RAPA and Wendantang （medium and high dose） groups showed lowered levels of IL-10 and TGF-β （P<0.01）， and the ORLI group showed down-regulated expression of TGF-β （P<0.01）. The expression of key molecules of the signaling pathway was up-regulated （P<0.05， P<0.01） while that of ULK1 was down-regulated （P<0.01） in all the intervention groups. Compared with the RAPA group， the Wendantang groups showed up-regulated expression of all autophagy marker proteins in adipocytes （P<0.01）. In addition， the low-dose Wendantang group showed elevated levels of inflammatory cytokines （except TNF-α） （P<0.05， P<0.01） and down-regulated expression of all key molecules of the signaling pathway （P<0.05， P<0.01）. The levels of inflammatory cytokines （except IL-16， MCP-1， and IL-10） were elevated in the medium-dose Wendantang group （P<0.05， P<0.01）. The expression of key molecules except PI3K of the signaling pathway was down-regulated in the medium- and high-dose Wendantang groups （P<0.05， P<0.01）. Compared with the ORLI group， low- and medium-dose Wendantang groups showed up-regulated expression of autophagy markers in adipocytes （P<0.01）， and the low-dose group showed elevated levels of inflammatory cytokines （IL-6， IL-4， and TGF-β） （P<0.01） and down-regulated expression of all key molecules of the signaling pathway （P<0.01）. The medium-dose Wendantang group showed up-regulated expression of IL-4 （P<0.01） and down-regulated expression of key molecules except PI3K of the signaling pathway （P<0.05， P<0.01）. The high-dose Wendantang group showed increased body mass， up-regulated expression levels of autophagy markers （ULK1， LC3 Ⅰ/Ⅱ） （P<0.05， P<0.01）， down-regulated expression of PIP3， mTORC1， and TSC1 （P<0.05， P<0.01）， and lowered levels of Beclin1， Atg5， TNF-α， and IL-13 （P<0.05， P<0.01）. ConclusionThe inflammation in obesity （syndrome of phlegm-dampness） is closely associated with the PI3K/Akt/mTOR pathway-mediated adipocyte autophagy. Wendantang can treat the chronic inflammation in obese rats with the syndrome of phlegm-dampness by regulating this signaling pathway and thus improve adipocyte autophagy.

Research progress on T cell glycolytic metabolism in oral lichen planus / 口腔疾病防治

@#Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa. The pathogenesis of OLP is still unclear. Immune abnormalities mediated by T cells and related cytokines play a crucial role in the pathogenesis of OLP. In recent years, glycolytic metabolism-related transporters, enzymes and regulators, such as glucose transporter-1 (Glut1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDHA), mammalian target of rapamycin (mTOR) and hypoxia inducible factor-1α (HIF-1a), have attracted an increasing amount of attention in OLP by regulating the proliferation and differentiation of T cells and the secretion of inflammatory factors. It has been shown that 2-deoxy-D-glucose (2-DG) or rapamycin (RAPA) inhibits the glycolytic metabolism of T cells and then inhibits OLP. This article reviews the research progress of glycolytic metabolism-related transporters, enzymes and regulatory factors in OLP in recent years.

Risk factors of new-onset hypertriglyceridemia in kidney transplant recipients: a single-center analysis / 器官移植

Objective To identify the risk factors of new-onset hypertriglyceridemia (HTG) in kidney transplant recipients. Methods Clinical data of 149 kidney transplant recipients were retrospectively analyzed. According to serum triglyceride (TG) level after operation, they were divided into the non-HTG group (TG≤1.7 mmol/L, n=60) and new-onset HTG group (TG>1.7 mmol/L, n=89). Baseline data of all recipients were compared between two groups. The risk factors of HTG in kidney transplant recipients were analyzed by generalized estimating equation (GEE), and validated by multiple regression equations. Results No significant differences were observed in baseline data between two groups (all P>0.05). Multivariate analysis showed that the incidence of HTG in the middle and high tacrolimus (Tac) concentration groups was higher than that in the low Tac concentration group [odds ratio (OR) 3.11, 95% confidence interval (CI) 1.22-7.93, P=0.018 in the middle Tac concentration group; OR 5.11, 95%CI 1.31-19.98, P=0.019 in the high Tac concentration group]. Compared with type-A blood recipients, the risk of new-onset HTG was significantly increased in type-O blood counterparts (OR 2.77, 95%CI 1.14-6.71, P=0.024). The risk of new-onset HTG was decreased along with the increase of preoperative globulin level (OR 0.93, 95%CI 0.87-0.99, P=0.043). At postoperative 3 months, Tac blood concentration in the new-onset HTG group was significantly higher compared with that in the non-HTG group, and significant difference was observed (P<0.05). Multiple regression equations confirmed that the risk of new-onset HTG in type-O blood kidney transplant recipients was higher than that in type-A blood counterparts, and the risk of new-onset HTG in the middle and high Tac concentration groups was higher than that in the low Tac concentration group (all P<0.05). Conclusions Type-O blood kidney transplant recipients are more prone to HTG. It is necessary to strengthen postoperative monitoring and control of blood lipids. The blood concentration of Tac probably affects the new-onset HTG in kidney transplant recipients. Maintaining an appropriate blood concentration of Tac may be beneficial to lowering the risk of HTG.

Pre-treatment of the beta3-adrenergic receptor agonist BRL37344 reduces in vivo myocardial ischemia/reperfusion injury by improving AMPK and SIRT1 activity and by suppressing mTOR and p70S6K signaling pathways

Abstract This study aimed to investigate the role and signaling pathways of β3-AR in myocardial ischemia/reperfusion (I/R) injury, which is one of the leading causes of death worldwide. 47 male rats were randomly divided into two main groups to evaluate infarct size and molecular parameters. Rats in both groups were randomly divided into 4 groups. Control (sham), I/R (30 min ischemia/120 min reperfusion), BRL37344 (BRL) (A) (5 µg/kg single-dose pre-treatment (preT) before I/R) and BRL (B) (5 µg/kg/day preT for 10 days before I/R). Infarct size was determined with triphenyltetrazolium chloride staining and analyzed with ImageJ program. The levels of AMPK, SIRT1, mTOR, and p70SK6 responsible for cellular energy and autophagy were evaluated by western blot. Infarct size increased in the I/R group (44.84 ± 1.47%) and reduced in the single-dose and 10-day BRL-treated groups (32.22 ± 1.57%, 29.65 ± 0.55%; respectively). AMPK and SIRT1 levels were decreased by I/R but improved in the treatment groups. While mTOR and p70S6K levels increased in the I/R group, they decreased with BRL preT. BRL preT protects the heart against I/R injury. These beneficial effects are mediated in part by activation of AMPK and SIRT1, inhibition of mTOR and p70S6K, and consequently protected autophagy.

Chinese Medicine Regulates mTOR Signaling Pathway to Prevent and Treat Osteoporosis： A Review / 中国实验方剂学杂志

Osteoporosis （OP）， a common systemic skeletal disease in the elderly， is characterised by bone loss and bone microstructural degeneration. Its clinical manifestations include increased bone fragility and bone pain. Furthermore， OP increases the risk of fracture due to the high bone fragility， which leads to lifelong disability or death， imposing a heavy economic and psychological burden on the patients and their families. The pathogenesis of OP is extremely complex and associated with a variety of factors such as proliferation and differentiation of osteoblasts， impairment of osteoclast activity and function， and abnormalities in autophagy activation. Recent studies have found that mammalian target of rapamycin （mTOR） signaing pathway is involved in the regulation of bone homeostasis， which can promote bone formation and improve bone metabolism and bone microstructure by regulating osteoblast proliferation and differentiation and osteoclast function and activating cellular autophagy， thus playing a crucial role in the prevention and treatment of OP. The prevention and treatment of OP with Chinese medicine has a long history， clear efficacy， multiple targets of action， low adverse effects， and wide medicine sources. Therefore， this paper briefly describes the role of mTOR signaling pathway in the development of OP by reviewing the latest research reports and summarizes in detail the latest research results on the treatment of OP with Chinese medicine extracts and prescriptions via the mTOR signaling pathway. This review aims to provide a basis for the in-depth research on the relationship between mTOR signaling pathway and OP and the clinical application of traditional Chinese medicine in the prevention and treatment of OP.

Mechanism of PI3K/Akt/mTOR Signaling Pathway Against Non-small Cell Lung Cancer and Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

Non-small cell lung cancer （NSCLC） is a malignant tumor of the respiratory system with a high incidence. The early symptoms are not typical， and most patients are diagnosed at an advanced stage， which seriously threatens the lives and health of people. Surgery， chemotherapy， and targeted therapy are the main means of treatment at present， but the consequent drug resistance and adverse reactions restrict these treatment methods with certain limitations. In recent years， with the development of traditional Chinese medicine （TCM） in tumor resistance， TCM has attracted extensive attention for its obvious therapeutic effect and fewer adverse reactions. Numerous signaling pathways are involved in the formation and development of NSCLC， where phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway is one of the key regulatory pathways. The PI3K/Akt/mTOR signaling pathway affects the proliferation， invasion， and metastasis of NSCLC cells by affecting the cycle， inhibiting the apoptosis， inhibiting the autophagy of tumor cells， and promoting tumor neovascularization. As revealed by the recent literature， Chinese medicine plays an indispensable role in NSCLC cell autophagy， cell cycle， apoptosis， invasion and metastasis， neovascularization， and reversal of drug resistance by regulating the PI3K/Akt/mTOR signaling pathway. Although some Chinese medicinal extracts or compounds have made great breakthroughs in some mechanisms of action in the treatment of NSCLC， these studies only remain at the level of in vitro cell experiments and animal studies in vivo. Researchers are faced with the great challenge of "transforming the research results of Chinese medicines into clinical applications". Based on the current research status in China and abroad， this paper reviewed Chinese medicine in the intervention in NSCLC through the regulation of PI3K/Akt/mTOR signaling pathway in recent years， in order to open up new ideas for NSCLC drug therapy research and also provide a useful reference for further mechanism research.