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.

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.

Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Huangqisan （HQS） in regulating autophagy to alleviate hepatic steatosis and improve non-alcoholic fatty liver disease （NAFLD） based on adenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodThe main chemical components and targets of HQS and NAFLD-related targets were collected from database and the intersection targets were used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network was constructed， and in vivo experimental verification was conducted. Sixty C57BL/6J male mice were randomly divided into normal control group （NCD）， model group high-fat diet （HFD）， metformin group （MET， 0.25 g·kg-1）， low-dose Huangqisan group （HQS-L， 0.5 g·kg-1）， and the high-dose Huangqisan group （HQS-H， 1 g·kg-1）， with 12 mice in each group after a one-week acclimatization period. NAFLD model was induced by HFD， and intragastric administration was performed at the same time， once a day for 13 weeks. Random blood glucose， serum total cholesterol （TC）， triglyceride （TG）， non-esterified fatty acid （NEFA）， low density lipoprotein-chdesterol （LDL-C） levels， and liver TG content were determined. The liver weight was weighed， and liver index was calculated. Hematoxylin-eosin （HE） staining， oil red O staining， transmission electron microscope （TEM）， real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot were used to verify the effect and reveal the potential mechanism of C57BL/6J mice in vivo. ResultThrough network pharmacology analysis， combined with previous studies， it was predicted that HQS may improve NAFLD by regulating autophagy via the AMPK/mTOR signaling pathway. The result of in vivo experiment showed that， as compared with NCD group， random blood glucose， body weight， serum TC， LDL-C， NEFA， liver weight， liver index， and liver TG content of mice in the HFD groups were significantly increased （P<0.01）. HE staining showed massive lipid droplets （LDs） vacuolated， oil red O staining showed lipid accumulation in liver cells， and no obvious autophagosomes and autolysosome were observed under TEM. The relative mRNA expression of LC3A、LC3B、AMPKα1 and protein expression of AMPK， phosphory phosphorylated（p）-AMPK， and p-AMPK/AMPK were significantly down-regulated （P<0.01）， while the protein expression of microtubule-associated protein 1 light chain 3 （LC3）Ⅱ/Ⅰ and p-mTOR was significantly up-regulated （P<0.01）. As compared with HFD groups， liver weight， serum TG， and NEFA levels in HQS-L and HQS-H groups were significantly deceased （P<0.05， P<0.01）. HE staining and oil red O staining showed the improvement of liver pathological changes after HQS administration. Under TEM， a small amount of autophagosome and autolysosome were observed. Besides， liver index was significantly decreased in the HQS-L group （P<0.01）， and random blood glucose， serum TC level and liver TG content were significantly decreased in the HQS-H group （P<0.05）. The results of Western blot and Real-time PCR showed that the mRNA expression of LC3A and LC3B and the protein expression of LC3Ⅱ/Ⅰ， p-AMPK， and p-AMPK/AMPK were significantly up-regulated （P<0.01）， while the mRNA expressions of p62 and protein expression of p62 and p-mTOR were significantly down-regulated （P<0.05， P<0.01）. ConclusionHQS may promote autophagy and restore autophagy flux via the AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.

Emodin Ameliorates High Glucose-Induced Podocyte Apoptosis via Regulating AMPK/mTOR-Mediated Autophagy Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro.@*METHODS@#MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.@*RESULTS@#HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation.@*CONCLUSION@#Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.

Genomic and epigenetic advances in malformations of cortical development / 中华神经科杂志

Malformations of cortical development (MCD) describe malformation lesions which are characterized by abnormal cortical structure or presence of heterotopic grey matter, sometimes associated with abnormal brain size. Recent progress in understanding the genetics and epigenetics in brain malformations has been driven by extraordinary advances in DNA sequencing technologies and DNA methylation profiling. For example, somatic mosaic mutations that activate mammalian target of rapamycin signaling in cortical progenitor cells are now recognized as the main cause of some types of MCD. In this review, the classification and genetic etiologies of MCD, especially focal cortical dysplasia, are summarized.

Effects of fluoride exposure on autophagy and AMPK/mTOR/ULK1 pathway in NG108-15 cells / 中华地方病学杂志

Objective:To investigate the effects of fluoride exposure on autophagy and the expression levels of adenosine monophosphate activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1) in mouse neuroblastoma and rat glioma fusion cells (NG108-15 cells).Methods:NG108-15 cells were cultured in vitro and divided into control group (0 mg/L), low fluoride group (20 mg/L), medium fluoride group (40 mg/L) and high fluoride group (80 mg/L) according to the final concentration of sodium fluoride, and the cells were collected after 24 h of treatment for standby. NG108-15 cells autophagy was detected by immunofluorescence/immunocytochemistry (IF/ICC method, the autophagy positive control group was treated with chloroquine phosphate); the mRNA expression levels of AMPK, mTOR and ULK1 in each group were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the protein expression levels of autophagy related protein microtubule-associated protein 1 light chain 3B (LC3B), AMPK, mTOR, ULK1, phosphorylation (p)-AMPK, p-mTOR, p-ULK1 in each group were detected by Western blotting. Results:No autophagosome was detected in the control group, and autophagosomes were detected in all the fluoride groups. The protein expression level of LC3B in the low, medium and high fluoride groups (1.80 ± 0.59, 2.16 ± 0.60, 2.30 ± 0.57) was significantly higher than that in the control group (1.00 ± 0.29, P < 0.05). The results of qRT-PCR showed that compared with the control group, the mRNA expression levels of AMPK in medium and high fluoride groups were higher (2.30 ± 0.57, 4.41 ± 1.05 vs 1.00 ± 0.01, P < 0.05); the mRNA expression levels of mTOR in the low, medium and high fluoride groups were lower (0.79 ± 0.04, 0.76 ± 0.09, 0.64 ± 0.10 vs 1.00 ± 0.01, P < 0.05), and the mRNA expression levels of ULK1 were higher (1.81 ± 0.39, 1.96 ± 0.35, 4.22 ± 1.03 vs 1.00 ± 0.01, P < 0.05). The results of Western blotting showed that compared with the control group, the protein expression levels of AMPK (1.21 ± 0.05, 1.20 ± 0.04, 1.30 ± 0.07 vs 1.00 ± 0.03), p-AMPK (1.12 ± 0.05, 1.20 ± 0.06, 1.49 ± 0.07 vs 1.00 ± 0.02), ULK1 (1.16 ± 0.05, 1.26 ± 0.05, 1.15 ± 0.05 vs 1.00 ± 0.04) and p-ULK1 (1.19 ± 0.04, 1.17 ± 0.02, 1.24 ± 0.05 vs 1.00 ± 0.05) in the low, medium and high fluoride groups were higher ( P < 0.05), and the protein expression levels of mTOR were lower (0.77 ± 0.03, 0.60 ± 0.03, 0.55 ± 0.04 vs 1.00 ± 0.04, P < 0.05); the protein expression levels of p-mTOR in the medium and high fluoride groups were lower (0.93 ± 0.05, 0.48 ± 0.02 vs 1.00 ± 0.02, P < 0.05). Conclusion:Fluoride exposure can induce autophagy in NG108-15 cells, and the expression of AMPK and ULK1 are up-regulated, while the expression of mTOR is down-regulated.

Research progress of the protection of rapamycin for proliferative vitreoretinopathy / 中华实验眼科杂志

Proliferative vitreoretinopathy (PVR) is a complication of ocular trauma, rhegmatogenous retinal detachment (RRD), and also a common cause of RRD repair surgery failure.Abnormal proliferation, migration and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play a leading role in the formation of PVR epiretinal membrane.Rapamycin is the specific inhibitor of mammalian target of rapamycin (mTOR). It selectively binds to the cell protein FKBP-12 and directly binds to the FKBP12-rapamycin domain (FRB) of FKBP rapamycin associated protein (FRAP) to inhibit mTOR activity.Rapamycin has a variety of rapalog (rapamycin analog), which inhibits cell proliferation and regulate cell cycle by inhibiting mTOR signal transduction pathway.It also plays a certain role in inhibiting RPE cell abnormal proliferation, migration and EMT in PVR, and protecting the repair of glial cells, inhibiting the inflammatory cells and preventing the vascular endothelial cell damage.In recent years, the clinical trials and drug studies have shown the important role of rapamycin in ocular diseases.In addition, the evidence on ocular administrations and drug safety of rapamycin has been gradually accumulated.This article reviewed the protective effects and safety of rapamycin on RPE cells and other cells in PVR.

Neonatal Smith-Kingsmore syndrome: case report and literature review / 中国新生儿科杂志

Objective:To study the clinical and genetic features of neonatal Smith-Kingsmore syndrome (SKS).Methods:The clinical data of a newborn with SKS admitted to our hospital in November 2021 were reviewed. Using "Smith-Kingsmore", "rapamycin gene", "newborn", "premature infant", "the mammalian target of rapamycin", "MTOR", "mTOR", "Smith-Kingsmore syndrome", "megalencephaly", "macrocephaly" and "hemimegalencephaly" as keywords, databases including CNKI, Wanfang Database, VIP database, PubMed, Embase, Web of Science and the Cochrane Library were searched from the date of establishment to January 1, 2022. The clinical and genetic features of neonatal SKS from published literature were summarized.Results:The case admitted to our hospital was a male preterm infant. The presenting symptoms were groan and hypotonia. The facial abnormalities included macrocrania, ocular hypertelorism, depressed nasal bridge and low-set ears. Brain MRI showed lateral ventricle enlargement. Whole-genome sequencing (WGS) showed mTOR gene nonsense heterozygous mutation (NM_004958.4:c.7255G>A:p.Glu2419Lys). Neither father nor mother had any pathogenic gene mutations. The infant had seizure at 2-month and phenobarbital was effective reducing seizure. Gross motor delay was present at 3-month. Sixteen related articles were retrieved, including eight articles with 10 neonatal cases. Among them, 6 cases were male. The main clinical features were megalencephaly or hemimegalencephaly (9/10), facial developmental malformation (8/10), hypotonia (6/10), large-for-gestational age (LGA) infants (5/10), cerebral ventricle dilation (4/10) and abnormal corpus callosum (4/10). All the gene mutations were missense mutations, including c.5395G>A(p.Glu1799Lys) mutation in 5 cases, c.4448G>T(p.Cys1483Phe) mutation in 1 case, c.4448G>T(p.Cys1483Tyr) mutation in 1 case, c.7235A>T(p.Asp2412Val) mutation in 1 case, c.5663T>G(p.Phe1888Cys) mutation in 1 case, c.5390C>T(p.Thr1799IIe) mutation in 1 case.Conclusions:The clinical phenotypes of neonatal SKS are diverse, including megalencephaly, facial malformation, LGA and hypotonia. The brain MR findings included (hemi) megalencephaly, cerebral ventricle dilation and corpus callosum hypoplasia. Most of the gene mutations are missense mutations and c.5395G>A(p.Glu1799Lys) is the hotspot.

Effect and Mechanism of Osthole on Proliferation and Apoptosis in Human Intrahepatic Cholangiocarcinoma HuCCT1 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of osthole on the proliferation and apoptosis in human intrahepatic cholangiocarcinoma HuCCT1 cells. MethodThe effect of 10， 20， 40， 80， and 120 μmol·L-1 osthole on the proliferation of HuCCT1 cells was detected by the cell counting kit-8 （CCK-8）. A blank group， and low-， medium-， and high-dose osthole groups （16， 32， and 64 μmol·L-1） were set up. The effect of osthole on cell clone formation rate was detected by colony formation assay. The effect of osthole on cell cycle and apoptosis was detected by flow cytometry. The effect of osthole on cell apoptotic morphology was detected by Hoechst 33342 fluorescent staining. The effect of osthole on cell cycle protein cyclin B1， proliferating cell nuclear antigen （PCNA）， cysteine-aspartic acid protease （Caspase）-9， Caspase-3， cleaved Caspase-9， cleaved Caspase-3， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， B-cell lymphoma-2 （Bcl-2）， phosphorylated protein kinase B （p-Akt）， phosphorylated mammalian target of rapamycin （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） was detected by Western blot. ResultThe cell viability in the osthole group（40，80，120 μmol·L-1） decreased （P<0.05，P<0.01）， with the half maximal inhibitory concentration （IC50） of 63.8 μmol·L-1 as compared with that in the blank group. Compared with the blank group， the osthole groups（32，64 μmol·L-1）showed reduced clone formation rate （P<0.01）， increased number of cells in the G2 phase （P<0.05，P<0.01）， decreased number of cells， increased pyknosis and fragmentation， increased apoptosis rate （P<0.05，P<0.01）， down-regulated expression of cyclin B1， PCNA， Bcl-2， Caspase-3， Caspase-9， p-Akt， p-mTOR， and p-RPS6 （P<0.05，P<0.01）， and up-regulated expression of cleaved Caspase-3， cleaved Caspase-9， and cleaved PARP （P<0.05，P<0.01）. ConclusionOsthole can inhibit the proliferation and promote the apoptosis of HuCCT1 cells， and its mechanism may be related to the Akt/mTOR signaling pathway.

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.

Effects of activating mTORC2/Akt signaling pathway on dopaminergic neurons and behaviors in 6-hydroxydopamin model mice / 解剖学报

Objective To explore the effect of activation of mammalian target of rapmycin complex 2(mTORC2)/Akt signaling pathway on dopaminergic neurons and behavior in 6-hydroxydopamine (6-OHDA) model mice and its possible mechanism. Methods Selecting 36 mice which The Nestin-CreERTM and ROSA26-LacZ reporter genes were detected at the same time in 3-month-old male C57BL/6J mice weighing 20-25 g divideng them into 4 gruops, NS+ corn oil group, 6-OHDA+corn oil group, 6-OHDA+PP242 group and 6-OHDA+A-443654 group, and 6-OHDA was injected into the right striatum of the brain to replicate the Parkinson’s disease (PD) model of mice, and then daily intraperitoneal injection of mTORC2/Akt signaling pathway agonist A-443654 or inhibitor PP242. Serum interleukin-1β (IL-1β) and tumor necrosis factor-α(TNF-α)levels were measured by enzyme-linked immunosorbent assay. Immunohistochemistry and immunofluorescence staining were performed to investigate the change of microglia, dopaminergic neurons as well as neural progenitor cells (NPCs). Western blotting was used to detect the expression of related protein of mTORC2/Akt signaling pathway including rictor, p-Akt and regulated in development and DNA dgmage responses 1(REDD1) and the interaction between them were verified by immunoprecipitation. Finally, the behavioral performance of each group of mice was observed. Results With the activation of microglia and the increase of inflammatory factors in PD model mice, the number of dopaminergic neurons in the substantia nigra(SN) decreased significantly, and the motor function of the mice was impaired, but the number of NPCs increased significantly compared with the control mice, mTORC2/Akt signaling pathway related protein expression was also significantly up-regulated. A-443654 treatment further up-regulated the expression of these proteins, meanwhile the indicators mentioned above were ameliorated. However, the inhibitor PP242 treatment group showed completely opposite result with the agonist group. Conclusion A-443654 can promote the proliferation of NPCs and the number of new-born dopaminergic neurons by up-regulating related proteins of mTORC2/Akt signaling pathway, and reducing the activation of microglia and the level of inflammation factors, which ultimately lead to the amelioration of SN-striatal dopaminergic neurons and behavioral performance in PD model mice.