Protein Expression Of p-mTOR Research Articles

Qihuang Jianpi Zishen Granules improves thrombocytopenia in mice with systemic lupus erythematosus by suppressing platelet autophagy via the Ca2+/CaMKK2/AMPK/mTOR signaling pathway

To explore the mechanism of Qihuang Jianpi Zishen Granules (QJZG) for improving thrombocytopenia in a mouse model of systemic lupus erythematosus (SLE). Twenty-four MRL/lpr lupus mice were randomized equally into 4 groups for treatment with daily gavage of saline, QJZG or prednisone (Pred) or intraperitoneal injection (twice a week) of CaMKK2 activator, with 6 C57BL/6 mice with saline gavage as the control group. After 8 weeks of treatment, the mice were examined for PLT, PCT, PDW, MPV, serum levels of TPO, IL-6, IL-10, TNF-α and IFN-γ, and calcium ion fluorescence intensity using ELISA or flow-through assay. RT-qPCR was used to detect platelet CaMKK2, AMPK2α, mTOR, Beclin1 and p62 mRNA expression levels, and the protein expressions of CaMKK2, p-CaMKK2, AMPK, p-AMPK, mTOR, p-mTOR, LC3, Beclin1 and p62 were detected using Western blotting. The saline-treated MRL/lpr lupus mice showed significantly lowered levels of PLT, PCT, IL-10, mTOR, p62 mRNA, p-mTOR and P62 with increased PDW, MPV, serum TPO, IL-6, TNF-α and IFN-γ levels, and platelet expressions of CaMKK2, AMPK, Bcl-1 mRNA, p-CaMKK2, p-AMPK, LC3II and Beclin1. These abnormalities were significantly improved in QJZG group and Pred group but worsened after treatment with the CaMKK2 activator. QJZG can ameliorate thrombocytopenia in mouse models of SLE by reducing inflammation and inhibiting platelet autophagy via regulating the Ca2+/CaMKK2/AMPK/mTOR signaling pathways.

Nek6 regulates autophagy through the mTOR signaling pathway to alleviate cerebral ischemia–reperfusion injury

ObjectiveCerebral ischemia–reperfusion injury (CIRI) is a major obstacle to neurological recovery after clinical treatment of ischemic stroke. The aim of this study was to investigate the molecular mechanism of Nek6 alleviating CIRI through autophagy after cerebral ischemia.Materials and methodsA mouse model of CIRI was constructed by middle cerebral artery occlusion (MCAO). TUNEL staining was used to observe the apoptosis of neuronal cells. The oxygen glucose deprivation/reoxygenation (OGD/R) model was established by hypoxia and reoxygenation. The cell apoptosis and activity was detected. Western blot was performed to detect the expression of autophagy-related proteins, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine 5’-monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway-related proteins. Cellular autophagy flux was observed by fluorometric method. NIMA-related kinase 6 (Nek6) mRNA stability was detected by actinomycin D treatment. Methylation RNA immunoprecipitation technique was used to detect Nek6 methylation level.ResultsNek6 expression was increased in both MCAO and OGD/R models. Overexpression of Nek6 in OGD/R inhibited apoptosis, decreased LC3II and Beclin-1 expression, increased p62 expression, and occurred lysosome dysfunction. Interference with Nek6 has opposite results. Nek6 overexpression promoted p-Akt and p-mTOR protein expressions, inhibited p-AMPK and p-UNC-51-like kinase 1 protein expressions and cell apoptosis, while LY294002, Rapamycin or RSVA405 treatment reversed this effect. Abnormal methyltransferase·like protein 3 (METTL3) expression in CIRI enhanced m6A modification and promoted Nek6 expression level.ConclusionThis study confirmed that Nek6 regulates autophagy and alleviates CIRI through the mTOR signaling pathway, which provides a novel therapeutic strategy for patients with ischemic stroke in the future.

Effect of electroacupuncture on neurological function in rats with cerebral ischemia-reperfusion injury based on AMPK/mTOR/ULK1 signaling pathway

To investigate the effect of electroacupuncture (EA) on neurological function in rats with cerebral ischemia-reperfusion injury (CIRI) by regulating adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) signaling pathway. Thirty-three male SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 11 rats in each group. The right middle cerebral artery occlusion/reperfusion (MCAO/R) model was prepared by thread occlusion method in the model group and the EA group. In the sham-operation group, no thread was inserted after vascular separation. After the success of modeling, in the EA group, EA was applied to "Baihui" (GV 20) and "Zusanli" (ST 36) on the affected side, with disperse-dense wave, the frequency of 2 Hz/15 Hz, for 20 min, once a day. EA was delivered continuously for 3 days. On day 1 and day 3 of operation, the score of the modified neurological deficit scale (mNSS) was evaluated. After intervention completion, the cerebral infarction area was measured by the thiazolyl blue tetrazolium chloride (TTC) method. Nissl staining was used to observe the damage of cortical neurons on the ischemic side in each group. Using transmission electron microscopy, the ultrastructure of cortical neurons on the ischemic side was observed. With the immunofluorescence method adopted, the positive expression of the related protein 1 light chain 3 (LC3) and benzyl chloroform (Beclin-1) on the ischemic side was detected. The protein expression of p-AMPK, AMPK, p-mTOR, mTOR, pS757-ULK1, ULK1 and chelating ligand 1 (p62) in the ischemic cortex was detected using Western blot method. ① Compared with the sham-operation group, in the model group, the mNSS score increased (P<0.01), the percentage of infarction area was increased (P<0.01); the cortical neurons on the ischemic side were loosely distributed, with karyopyknosis and vacuolization, and the number of neurons was reduced (P<0.01); the cells were swollen and ruptured, mitochondrial shrunk, electron density higher, and there were a large number of autophagic debris. The positive expression of LC3 and Beclin-1 was elevated (P<0.01), and p-mTOR/mTOR, pS757-ULK1/ULK1 and the protein expression of p62 dropped (P<0.01) in the ischemic cortex. ② Compared with the model group, in the EA group, the mNSS score was reduced (P<0.05). The percentage of cerebral infarction area was descreased (P<0.01); and the neurons were regularly distributed, the number of neurons increased (P<0.01), the structure of mitochondria was clearer, the crest fracture alleviated, and the damage of neurons attenuated. The positive expression of LC3 and Beclin-1 was dropped (P<0.01), and p-AMPK/AMPK reduced (P<0.05), and p-mTOR/ mTOR, pS757-ULK1/ULK1 and the protein expression of p62 increased (P<0.01) in the ischemic cortex. EA at "Baihui" (GV 20) and "Zusanli" (ST 36) inhibits autophay, attenuates neurological deficit and cerebral pathological damage in CIRI rats to protect the nerves, which may be obtained by regulating AMPK/mTOR/ULK1 signaling pathway.

The Role of Serine Protease 8 in Mediating Gefitinib Resistance in Non-small Cell Lung Cancer.

This investigation aims to explore the expression levels of serine protease 8 (PRSS8) in gefitinib-resistant Non-Small Cell Lung Cancer (NSCLC) cell lines (PC9/GR) and elucidate its mechanism of action. We measured PRSS8 expression in gefitinib-resistant (PC9/GR) and sensitive (PC9) NSCLC cell lines using Western blot analysis. PRSS8-specific small interfering RNA (PRSS8-siRNA), a recombinant plasmid, and a corresponding blank control were transfected into PC9/GR cells. Subsequently, Western blot analyses were conducted to assess the expression levels of PRSS8, phosphorylated AKT (p-AKT), AKT, phosphorylated mTOR (p-mTOR), mTOR, and various apoptosis-related proteins within each group. Additionally, a cell proliferation assay utilizing Cell Counting Kit-8 (CCK8) was performed on each group treated with gefitinib. PRSS8 expression was markedly higher in PC9/GR cells compared to PC9 cells (p < 0.05). The group treated with PRSS8-siRNA exhibited significantly reduced protein expression levels of PRSS8, p-AKT, p-mTOR, β-catenin, and BCL-2 compared to the control siRNA (Con-siRNA) group, whereas expressions of Caspase9 and Bax were significantly increased. In the untransfected PC9/GR cells, protein expressions of PRSS8, p-AKT, pmTOR, and BCL-2 were significantly elevated when compared with the plasmid-transfected group, which also showed a significant reduction in Bax expression. The proliferative activity of the PRSS8-siRNA group postgefitinib treatment was significantly diminished at 24, 48, and 72 hours in comparison to the Con-siRNA group. The findings indicate that PRSS8 contributes to the acquisition of resistance to gefitinib in NSCLC, potentially through regulation of the AKT/mTOR signaling pathway.

Integrated network pharmacology, metabolomics and molecular docking analysis to reveal the mechanisms of quercetin in the treatment of hyperlipidemia

Hyperlipidemia (HLP) is a significant contributor to cardiovascular diseases. Quercetin (QUE), a naturally occurring flavonoid with diverse bioactivities, has garnered attention due to its potential therapeutic effects. However, the precise mechanisms underlying the effects of QUE on HLP remain unclear. In this study, an ultra-high-performance liquid chromatography-quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive-MS) metabolomics strategy was employed to obtain metabolite profiles, and potential biomarkers were identified following data analysis. Network pharmacology and Drug Affinity Responsive Target Stability (DARTS) assays were utilized to explore the potential targets of QUE for HLP treatment. The results of metabolomics and network pharmacology were then integrated to identify the key targets and metabolic pathways involved in the therapeutic action of the QUE against HLP. Molecular docking and experimental validation were performed to confirm these key targets. A comprehensive database search identified 138 QUE-HLP-related targets. A protein-protein interaction (PPI) network was constructed using STRING, and the shared targets were filtered with Cytoscape. Among these, AKT1, TNF, VEGFA, mTOR, SREBP1, and SCD emerged as potential therapeutic targets. These findings were validated using in vitro cell experiments. Additionally, the mechanism of action of QUE against HLP was evaluated by integrating network pharmacology with metabolomics, identifying two metabolomic pathways crucial to HLP treatment. DARTS experiments confirmed the stable binding of QUE to FASN, p-mTOR, SREBP1, and p-AKT. In HepG2 cells treated with palmitic acid (PA), QUE significantly reduced the mRNA expression of ACLY, ACACA, FASN, and SCD (p < 0.05). Western blot analysis revealed that PA significantly increased protein expression of p-mTOR, SREBP1, FASN, and p-AKT (p < 0.05). In summary, our study provides novel insights into the protective mechanisms of QUE against HLP and offers valuable information regarding its potential benefits in clinical treatment.

Ling gui shen fu decoction ameliorates cardiac injury in ischemic heart disease rats by regulating ANP32A/HIF2α/HMGB1 signal route

Purpose: To investigate the effect of ling gui shen fu decoction (LGSFD) on cardiac injury and cardiomyocyte apoptosis in rats with ischemic heart disease (IHD), and to elucidate the underlying molecular mechanism. Methods: An IHD rat model was established by performing coronary artery occlusion surgery on the left anterior descending (LAD) of rats. The rats were assigned equally to 5 groups: sham-operated group (sham group), IHD group, IHD + perindopril group, IHD + low- dose LGSFD group (IHD + LGSFD-L group), and IHD + high-dose LGSFD group (IHD+LGSFD-H group). The LGSFD was given via gavage. A hypoxia-induced cardiomyocyte model was established by subjecting H9C2 cells to hypoxia. Then, LGSFD-containing serum or blank serum was used to treat the hypoxic rat cardiomyocytes (H9C2). The severity of cardiac injury and extent of apoptotic changes in cardiomyocytes was determined using hematoxylin-eosin (H&amp;E) staining and TUNEL staining, while immunoblotting was used to measure the protein expressions of ANP32A, p-AKT, AKT, HIF-2α, p-mTOR, mTOR and HMGB1. Results: The cardiac injury of rats in the LGSFD groups was significantly reversed, relative to the IHD group (p &lt; 0.05). Moreover, LGSFD reduced the level of apoptosis in hypoxia- induced H9C2 cells and upregulated the concentrations of ANP32A, p-AKT, HIF-2α and p-mTOR. However, the expression levels of HMGB1 were decreased in the heart tissues of IHD rats and hypoxic H9C2 cells. Silencing of ANP32A with ANP32A siRNA (siANP32A) down- regulated ANP32A, p-AKT, HIF-2α and p-mTOR, but up-regulated apoptosis and expression levels of HMGB1 in hypoxic H9C2 cells. Conclusion: LGSFD ameliorates cardiac injury in IHD rats by inhibiting cardiomyocyte apoptosis via the ANP32A/HIF-2α/HMGB1 axis. Further investigations are recommended into the specific mechanism of LGSFD effect using clinical samples, in order to facilitate its clinical development.

Extracorporeal Shock Wave and Melatonin Alleviate Joint Capsule Fibrosis after Knee Trauma in Rats by Regulating Autophagy.

Joint contracture is a common clinical problem affecting joint function. Capsule fibrosis plays a pivotal role in the progression of Joint contracture. Previous studies have reported that autophagy plays a regulatory role in visceral fibrosis. This study aimed to investigate whether extracorporeal shock wave therapy (ESWT) and melatonin alleviate joint capsule fibrosis in rats with extended knee joint contracture by regulating autophagy. A rat knee joint extension contracture model was made. Then, the rats were treated with ESWT, melatonin, ESWT + melatonin, or ESWT + melatonin + mTOR agonist for 4 weeks. The range of motion (ROM) of the knee joints was measured. Joint capsules were collected and observed for pathological changes by H&E and Masson staining. LC3B protein expression was evaluated by immunofluorescence staining. TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ, LC3, ATG7, Beclin1, p-AMPK, p-mTOR and p-ULK1 protein expressions were measured by Western blot assay. The intervention groups had significantly improved ROM of knee joint (P < 0.05), significantly improved pathological changes on HE and Masson staining, significantly decreased protein expressions of TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ and pmTOR (P < 0.05), and significantly increased protein expressions of LC3B, LC3II/LC3I ratio, ATG7, Beclin1, p-AMPK, and p-ULK1 (P < 0.05). Among these groups, the effects demonstrated by the ESWT + melatonin group were the best. With the mTOR agonist supplement, the therapeutic effects of extracorporeal shock waves and melatonin were significantly reduced. ESWT plus melatonin alleviated knee joint capsule fibrosis in rats by regulating autophagy.

Tangningtongluo Tablet ameliorates pancreatic damage in diabetic mice by inducing autophagy and inhibiting the PI3K/Akt/mTOR signaling pathway

BackgroundDiabetes is a metabolic disease characterized by hyperglycaemia. Tangningtongluo Tablet (TNTL) is an inpatient formula extensively utilized to treat diabetes mellitus (DM), but the protective mechanism is not clear. This study aimed to investigate the relevant mechanisms by which TNTL affects pancreatic damage in diabetic mice and autophagy. MethodsThe impact of TNTL on pancreatic damage in diabetic mice in vitro and in vivo was investigated via glucose and lipid metabolism analyses, HE staining, CCK-8, TUNEL staining, Annexin V/PI, and Western blotting. Molecular docking and Western blotting were used to verify the results of network pharmacological analysis, which was carried out to explore the mechanism by which TNTL affects DM. The autophagosome levels were visualized via RFP-GFP-LC3 and transmission electron microscopy, and lysosomal function was evaluated via Lysotracker red staining. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the expression of the autophagy proteins LC3, p62 and LAMP2. ResultsCompared with the model group, TNTL protected pancreas from oxidative stress, decreased the level of MDA, increased the levels of SOD and GSH-px, induced the occurrence of autophagy and decreased the levels of apoptotic factors. Moreover, TNTL inhibited the protein expression of p-PI3K, p-Akt and p-mTOR, increased the levels of LC3 and LAMP2 and decreased the level of p62, and the autophagy inhibitor CQ blocked the protective effect of TNTL on pancreatic damage in diabetic mice. ConclusionThese results demonstrated that TNTL ameliorated pancreatic damage in diabetic mice by inhibiting the PI3K/Akt/mTOR signaling and regulating autophagy.

Dapagliflozin restores autophagy and attenuates apoptosis via the AMPK/mTOR pathway in diabetic nephropathy rats and high glucose-induced HK-2 cells.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus. Significantly reduced levels of autophagy in diabetic kidneys play an important role in the development of DN. The present study investigated the effects of dapagliflozin (DAP) on renal autophagy and AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in vivo and in vitro. We explored the effect of DAP in streptozotocin (STZ)-induced DN rats. The anti-DN effect of DAP was assessed by body weight, kidney weight/body weight ratio, blood and urine biochemical parameters, and pathological changes of kidney tissue. Number of autophagosomes in the kidney was investigated through Transmission electron microscopy. Besides, cell viability and apoptosis of DAP alone or combined with Compound C (CC, a selective AMPK inhibitor)-treated high glucose (HG)-induced HK-2 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry assays. Immunohistochemistry, Western blot, Enzyme-linked immunosorbent assay (ELISA), and immunofluorescence were employed to detect the expression levels of extracellular matrix (ECM) deposition, autophagy, apoptosis, and AMPK/mTOR pathway-associated targets in vivo and in vitro. The results showed that DAP ameliorated the body weight and decreased kidney weight, fasting blood glucose, and serum/urine biochemical parameters of renal damage, as well as renal pathological changes. Moreover, DAP significantly ameliorated HG-induced cell apoptosis and ECM deposition in HK-2 cells. However, these favorable effects of DAP could be abolished by co-treatment with CC in HG-induced HK-2 cells. Mechanistically, DAP can enhance autophagy in DN including increased LC3-II/I ratio, Beclin-1, p-AMPK protein levels, and decreased p62 and p-mTOR protein expressions, as well as inhibited renal fibrosis and apoptosis. In summary, DAP alleviated fibrosis, apoptosis, and autophagy in DN rats and HG-induced HK-2 cells by regulating the AMPK/mTOR pathway.

The 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole (STT) Inhibits Liver Cancer Cell Growth by Targeting Akt/mTOR Pathway.

The present study was aimed to investigate the proliferation inhibitory ability of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on SNU449 and Huh7 cells. Moreover, the mechanism associated with the suppression of liver cancer cell proliferation by STT was also studied. The results revealed that STT suppresses proliferation of SNU449 and Huh7 cells to 28 and 21%, respectively treatment with 20 µM. The clonogenic survival of SNU449 and Huh7 cells was also significantly reduced after incubation with STT compared to the control cultures. In comparison to the control, STT treatment significantly decreased the invasive potential of SNU449 cells. Treatment with STT led to a prominent suppression in p62 and increase in LC3B protein expression in SNU449 cells compared to the control cells. The STT treatment dramatically decreased p-Akt and p-mTOR protein expression in SNU449 cells. Docking study revealed that STT interacts via traditional hydrogen bonding with the glutamine, phenylalanine, leucine, serine, arginine, aspartic acid, and lysine residues of Akt protein. In summary, the current study demonstrates that STT effectively suppresses the viability of SNU449 and Huh7 liver cancer cells. Moreover, STT treatment of the liver cancer cells also significantly reduces the clonogenic survival and invasive potential of SNU449 cells. Treatment of liver cancer cells with STT increases the expression of autophagic, targets anti-autophagic protein expression and down-regulates Akt/mTOR pathway to inhibit cancer growth and proliferation. Thus, STT exhibits prominent anticancer effect and needs to be investigated further as a potential candidate for the treatment of liver cancer.

Effects of maternal hawthorn-leaf flavonoid supplementation on the intestinal development of offspring chicks

Metabolic disorders in maternal generation during the late egg-laying period have adverse effects on neonatal development. The study was conducted to clarify the effects of maternal feeding of hawthorn-leaf flavonoid (HF) on the microbial community and intestinal development of chicks. Breeder hens were fed a basic corn-soybean diet, while the treatment groups were supplemented with 30 or 60 mg/kg HF. The offspring chicks were divided into CON, LHF, and HHF groups according to the maternal treatments. Maternal HF supplementation at 60 mg/kg increased the average daily gain and decreased the feed conversion rate of chicks (P < 0.05), but did not affect the average daily feed intake. HF treatments increased the villus height to crypt depth ratio and up-regulated the protein expressions of PCNA, IGF-1R, PI3K and p-mTOR in the jejunum (P < 0.05) of 1-day-old and 14-day-old chicks. Additionally, maternal HF treatment up-regulated the mRNA expression of tight junction transmembrane proteins (occludin) and scaffolding proteins (ZO-1 and ZO-2) in the jejunum of 1-day-old chicks (P < 0.05). Moreover, the maternal effects of HF on ZO-1 expression could last for 14 d (P < 0.05). Interestingly, dietary HF supplementation altered the vertically transmitted microbial community from breeder hens to chicks, especially increased the relative abundance of probiotics (i.e., Clostridium_sensu_stricto_1) in the meconium of chicks (P < 0.05), which may help with early gut microbiota colonization and intestinal development. In summary, dietary HF supplementation for breeder hens altered the bacterial community of neonates and might promote intestinal development of chicks through the IGF-1R/AKT/mTOR signaling pathway.

Repositioning of baricitinib for management of memory impairment in ovariectomized/D-galactose treated rats: A potential role of JAK2/STAT3-PI3K/AKT/mTOR signaling pathway

AimsNeuroinflammation plays a pivotal role in amyloid β (Aβ) plaques formation which is among the hallmarks of Alzheimer's disease (AD). The present study investigated the potential therapeutic effects of baricitinib (BAR), a selective JAK2/ STAT3 inhibitor, in ovariectomized/ D-galactose (OVX/D-gal) treated rats as a model for AD. Main methodsTo induce AD, adult female rats (130–180 g) underwent bilateral ovariectomy and were injected daily with 150 mg/kg, i.p. D-gal for 8 consecutive weeks. BAR (10 and 50 mg/kg/day) was then given orally for 14 days. Key findingsBAR in a dose-dependent effect mitigated OVX/D-gal-induced aberrant activation of JAK2/STAT3 signaling pathway resulting in significant decreases in the expression of p-JAK 2, and p-STAT3 levels, along with deactivating AKT/PI3K/mTOR signaling as evidenced by deceased protein expression of p-AKT, p-PI3K, and p-mTOR. As a result, neuroinflammation was diminished as evidenced by decreased NF-κβ, TNF-α, and IL-6 levels. Moreover, oxidative stress biomarkers levels as iNOS, and MDA were reduced, whereas GSH was increased by BAR. BAR administration also succeeded in reverting histopathological alterations caused by OVX/D-gal, increased the number of intact neurons (detected by Nissl stain), and diminished astrocyte hyperactivity assessed as GFAP immunoreactivity. Finally, treatment with BAR diminished the levels of Aβ. These changes culminated in enhancing spatial learning and memory in Morris water maze, and novel object recognition test. SignificanceBAR could be an effective therapy against neuroinflammation, astrogliosis and cognitive impairment induced by OVX/ D-gal where inhibiting JAK2/STAT3- AKT/PI3K/mTOR seems to play a crucial role in its beneficial effect.

Anemoside B4 attenuates necrotic enteritis of laying hens induced by Clostridium perfringens via inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways

Poultry necrotic enteritis is an important enteric disease which might be controlled by antibiotics. However, with the excessive use of antibiotics, the phenomenon of drug resistance of Clostridium perfringens is becoming increasingly prominent. Anemoside B4 exhibits important anti-inflammatory, antioxidant and immunomodulatory effects. This study was performed to estimate the effect of Anemoside B4 on chicken necrotic enteritis induced by C. perfringens in vivo and in vitro. In the in vivo experiment we investigated the efficacy of Anemoside B4 on the growth curve, biofilm formation, haemolytic activity, virulence-related gene expression and NF-κB and PI3K/AKT/mTOR activation in Caco-2 cells induced by C. perfringens. The results showed that 12.5–50 μg/mL Anemoside B4 had no antibacterial activity but could inhibit biofilm formation, attenuate haemolytic activity and virulence-related gene expression of C. perfringens and weaken NF-κB and PI3K/Akt/mTOR activation triggered by C. perfringens in Caco-2 cells. In the in vivo experiment, 60 17-day-old healthy White Leghorns were randomly divided into six groups. The growing laying hens of the control group were fed a basic diet, and those of the five challenged groups were fed a basic diet (infection group), added 0.43 g/kg Anemoside B4 (0.43 g/kg Ane group), 0.86 g/kg Anemoside B4 (0.86 g/kg Ane group), 1.72 g/kg Anemoside B4 (1.72 g/kg Ane group) and 40 mg/kg lincomycin (lincomycin group), respectively. All challenged laying hens were infected with 1 × 109 CFU C. perfringens from day 17–20. Blood and intestinal samples were obtained, and the data demonstrated that Anemoside B4 improved the blood biochemical parameters, attenuated jejunum tissue injury, increased the spleen, thymus, bursa of fabricius index, and decreased lesion scores of the jejunum and the ileum. In the jejunum, Anemoside B4 and lincomycin downregulated the expression of IL-1β, IL-6, IL-10, TNF-α and IFN-γ at mRNA levels. Moreover, Anemoside B4 significantly enhanced both mRNA and protein levels of tight junctions ZO-1, Claudin-1 and MUC-2 in the jejunum. Anemoside B4 weakened p-P65, p-PI3K, p-Akt and p-mTOR protein expression in the jejunum infected by C. perfringens. Diets supplemented with Anemoside B4 alleviated C. perfringens-induced necrotic enteritis in laying hens by inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways and improving intestinal barrier functions.

Aminophylline suppresses chronic renal failure progression by activating SIRT1/AMPK/mTOR-dependent autophagy.

Chronic renal failure (CRF) is a severe syndrome affecting the urinary system for which there are no effective therapeutics. In this study, we investigate the effects and mechanisms of aminophylline in preventing CRF development. A rat model of chronic renal failure is established by 5/6 nephrectomy. The levels of serum creatinine (SCR), urinary protein (UPR), and blood urea nitrogen (BUN) are detected by ELISA. Histological evaluations of renal tissues are performed by H&E, Masson staining, and PAS staining. Functional protein expression is detected by western blot analysis or immunofluorescence microscopy. Glomerular cell apoptosis is determined using the TUNEL method. Results show that Aminophylline significantly reduces the levels of SCR, UPR, and BUN in the CRF model rats. Histological analyses show that aminophylline effectively alleviates renal tissue injuries in CRF rats. The protein expression levels of nephrin, podocin, SIRT1, p-AMPK, and p-ULK1 are greatly increased, while p-mTOR protein expression is markedly decreased by aminophylline treatment. Additionally, the protein level of LC3B in CRF rats is significantly increased by aminophylline. Moreover, aminophylline alleviates apoptosis in the glomerular tissues of CRF rats. Furthermore, resveratrol promotes SIRT1, p-AMPK, and p-ULK1 protein expressions and reduces p-mTOR and LC3B protein expressions in CRF rats. Selisistat (a SIRT1 inhibitor) mitigates the changes in SIRT1, p-AMPK, p-ULK1, p-mTOR, and LC3B expressions induced by aminophylline. Finally, RAPA alleviates renal injury and apoptosis in CRF rats, and 3-MA eliminates the aminophylline-induced inhibition of renal injury and apoptosis in CRF rats. Aminophylline suppresses chronic renal failure progression by modulating the SIRT1/AMPK/mTOR-mediated autophagy process.

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway

BackgroundAcute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis.ObjectiveThis study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism.MethodsBlood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis.ResultsInterleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis.ConclusionThis study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

3-Hydroxy-3-methylglutaryl-CoA synthase 2 facilitates erectile dysfunction via inhibiting autophagy by enhancing the mammalian target of rapamycin pathway in type 1 diabetic mellitus rats.

The relationship between erectile dysfunction (ED) and type 1 diabetes mellitus (T1DM) is currently a hot topic of medical research. It has been reported that autophagy plays a crucial role in causing erectile dysfunction in T1DM. Recent research has shown that mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) is strongly linked to the development of T1DM. However, the specific mechanism by which it regulates the erectile function is not yet fully understood. To investigate whether HMGCS2 affects erectile function in type 1 diabetic rats by regulating autophagy in corpus cavernosum endothelial cells (CCECs). First, the rat model of T1DM was established. Then, the ratio of maximum penile intracavernous pressure (ICPmax) and mean arterial pressure (MAP) was detected to assess the erectile function in various groups, and the protein expression of HMGCS2, mTOR and p-mTOR was evaluated by western blot (WB) and immunohistochemistry (IHC). To explore the relationship between HMGCS2 and the mTOR signaling pathway in T1DM ED rats, we silenced the expression of HMGCS2 and activated the mTOR signaling pathway with MHY1485 in CCECs and then assessed the expression of beclin1, P62, LC3, autophagosome, endothelial nitric oxide synthase (eNOS), phosphorylation of eNOS (p-eNOS), and nitric oxide (NO) to evaluate autophagy and the erectile function by reverse transcription quantitative polymerase chain reaction and western blot. The study conducted on T1DM ED rats showed that the expression of HMGCS2 was significantly increased, while the autophagy was suppressed. Additionally, the mTOR signaling pathway was highly activated. In contrast, when HMGCS2 was silenced in vitro, p-mTOR/mTOR was reduced, and autophagy was improved. These effects were accompanied by the enhanced activity of eNOS. Furthermore, when HMGCS2 was silenced and the mTOR signaling pathway was simultaneously activated, the results revealed a decrease in autophagy as well as a reduction in activity of eNOS in comparison to just silencing HMGCS2 alone. HMGCS2 upregulation in T1DM rats inhibited autophagy and eNOS activity by activating the mTOR pathway and led to a decrease in the erectile function.

Ginsenoside Rg1 Induces Autophagy in Colorectal Cancer through Inhibition of the Akt/mTOR/p70S6K Pathway.

This study aimed to elucidate the anti-colon cancer mechanism of ginsenoside Rg1 in vitro and in vivo. Cell viability rate was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay. The inhibitory effect of ginsenoside Rg1 against CT26 cell proliferation gradually increased with increasing concentration. The in vivo experiments also demonstrated an antitumor effect. The monodansylcadaverine (MDC), transmission electron microscopy (TEM), and expression of autophagy marker proteins confirmed that ginsenoside Rg1 induced autophagy in vitro. Ginsenoside Rg1 induced autophagy death of CT26 cells, but this effect could be diminished by autophagy inhibitor (3-methyladenine, 3-MA). Additionally, in a xenograft model, immunohistochemical analysis of tumor tissues showed that the LC3 and Beclin-1 proteins were highly expressed in the tumors from the ginsenoside Rg1-treated nude mice, confirming that ginsenoside Rg1 also induced autophagy in vivo. Furthermoer, both in vivo and in vitro, the protein expressions of p-Akt, p-mTOR, and p-p70S6K were inhibited by ginsenoside Rg1, which was verified by Akt inhibitors. These results indicated that the mechanism of ginsenoside Rg1 against colon cancer was associated with autophagy through inhibition of the Akt/mTOR/p70S6K signaling pathway.

Effects of dietary citric acid on growth performance, mineral status, body and muscle composition, muscle growth and mTOR signaling in yellow catfish Pelteobagrus fulvidraco fed with low-manganese diets

The positive impacts of dietary citric acid (CA) on aquaculture species have been studied. However, the wide application of CA in aquatic-feed requires a comprehensive understanding of its nutritional functions. Here, an 8- week feeding trial was performed to determine whether dietary CA supplementation could improve manganese (Mn) utilization and thus reduce Mn excretion by investigating the effect of dietary CA on growth performance, mineral statues, body and muscle composition, muscle growth and mTOR signaling of yellow catfish Pelteobagrus fulvidraco fed with low-manganese (L-Mn) diets. Four isonitrogenous and isolipidic diets were formulated and fed to triplicated groups of fish. A suitable Mn diet containing 5.7 mg kg−1 Mn was set as the control, while the diets without extra Mn addition was regarded as L-Mn group. The other two diets were supplemented with 30 g kg−1 CA into the control and L-Mn diet, respectively. The results showed that the weight gain, specific growth rate, feed efficiency, protein content in muscle, iron concentration in serum, and Mn content in whole body and muscle were significantly decreased after L-Mn diet and resumed by dietary CA addition (P < 0.05). Dietary low-Mn markedly decreased the diameters of muscle fibers, which was alleviated by CA supplementation. Dietary Mn and CA interacted to affect the density of the muscle fiber density, the mRNA expression of muscle development-, myocyte myogenic and myocyte enhancer factors-related genes (P-interaction <0.05). Besides, expression of genes in muscle involved in mTOR signaling including mtor, s6 and eif4e were significantly down-regulated in fish fed L-Mn diet compared to the control. CA supplementation reversed the L-Mn diet induced decrease of their mRNA levels. Furthermore, the protein expression of p-mTOR, S6 and p-S6 were significantly decreased after L-Mn diet, and were upregulated by CA supplementation in the muscle (P < 0.05). Taken together, this study revealed that CA as a feed additive could improve growth performance, mineral bioavailability, muscle growth and protein synthesis in yellow catfish under the condition of Mn deficiency. These positive effects may be partially attributed to dietary CA supplementation improving Mn absorption and bioavailability. This study also has a significance for the development of environmentally friendly feeds for the aquatic species.

Sestrin 2 protects human lens epithelial cells from oxidative stress and apoptosis induced by hydrogen peroxide by regulating the mTOR/Nrf2 pathway.

We aimed to explore the effect and potential mechanism of Sestrin 2 (SESN2) in human lens epithelial cells (HLECs). To mimic the oxidative stress environment, SAR01/04 cells were treated with 200μM hydrogen peroxide (H2O2) for 24h. Cell viability and apoptosis were checked by cell counting kit-8 and flow cytometry. Western blot was taken to check the protein changes of SESN2, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, ribosomal protein S6 kinase B1 (p70S6K), p-p70S6K, and nuclear factor erythroid 2-related factor 2 (Nrf2). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected via the corresponding reagent kit. The levels of interleukin (IL)-1β, IL-18, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. SESN2 was down-regulated in cataract lens tissue and up-regulated in SAR01/04 cells treated with H2O2. Under treatment of H2O2, up-regulation of SESN2 improved cell viability, enhanced the activity of SOD and CAT, inhibited cell apoptosis, and reduced the levels of MDA, ROS, IL-1β, IL-18, and TNF-α, while down-regulation of SESN2 caused the contrary effects. Further bioinformatics analysis suggested that SESN2 regulated the mTOR signaling pathway. Treatment of H2O2 inhibited p-mTOR and p-p70S6K protein expression, while overexpression of SESN2 increased p-mTOR and p-p70S6K protein expression in the H2O2 group and down-regulation of SESN2 further decreased p-mTOR and p-p70S6K protein expression in the H2O2 group. Additionally, H2O2 increased Nrf2 protein expression, and overexpression of SESN2 further increased Nrf2 protein expression in the H2O2 group. Importantly, rapamycin (an inhibitor of mTOR signaling pathway) and knockdown of Nrf2 reversed the promotive effects of SESN2 on cell viability and the inhibitive effects of SESN2 on cell apoptosis, oxidative stress, and inflammatory reaction. SESN2 protected HLECs damage induced by H2O2, which was related to the activation of mTOR/Nrf2 pathway.

Ochratoxin A-enhanced glycolysis induces inflammatory responses in human gastric epithelium cells through mTOR/HIF-1α signaling pathway

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide with potential nephrotoxic, hepatotoxic and carcinogenic effects. We previously showed for the first time that OTA treatment enhanced glycolysis in human gastric epithelium (GES-1) cells in vitro. Here, we found that OTA exposure activated inflammatory responses, evidenced by increasing of NF-κB signaling pathway-related protein (p-p65 and p-IκBα) expressions and elevating of inflammatory cytokine (IL-1β and IL-6) mRNA expressions in GES-1 cells. To elucidate the role of glycolysis in inflammatory effects triggered by OTA, we pretreated GES-1 cells with glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) before OTA exposure. The result showed that 2-DG reduced the protein expressions of p-p65 and p-IκBα and alleviated the mRNA expressions of inflammatory cytokines in OTA-treated GES-1 cells. Furthermore, OTA activated the mTOR/HIF-1α pathway by increasing the protein expressions of p-mTOR, p-eIF4E and HIF-1α, and inhibition of mTOR with rapamycin or silencing HIF-1α with siRNA significantly attenuated OTA-enhanced glycolysis by reducing glycolysis related genes and thereby decreasing inflammatory effects of GES-1 cells. These results demonstrate that OTA activates inflammatory responses in GES-1 cells and this is controlled by mTOR/HIF-1α pathway-mediated glycolysis enhancement. Our findings provide a novel mechanistic view into OTA-induced gastric cytotoxicity.