High-dose Vitamin C injection ameliorates against sepsis-induced myocardial injury by anti-apoptosis, anti-inflammatory and pro-autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR signaling pathways in rats.

AIMS: This study aimed to evaluate the effects of VC on SIMI in rats. METHODS: In this study, the survival rate of high dose VC for SIMI was evaluated within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and high dose VC (500 mg/kg i.v.) group. The animals in each group were treated with drugs for 1 day, 3 days or 5 days, respectively. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6, IL-10 and TNF-α) in serum were measured using ELISA kits. Western blot was used to detect proteins related to apoptosis, inflammation, autophagy, MAPK, NF-κB and PI3K/Akt/mTOR signaling pathways. RESULTS: High dose VC improved the survival rate of SIMI within 7 days. Echocardiography, HE staining and myocardial enzymes showed that high-dose VC relieved SIMI in rats in a time-dependent manner. And compared with CLP group, high-dose VC decreased the expressions of pro-apoptotic proteins, while increased the expression of anti-apoptotic protein. And compared with CLP group, high dose VC decreased phosphorylation levels of Erk1/2, P38, JNK, NF-κB and IKK α/ß in SIMI rats. High dose VC increased the expression of the protein Beclin-1 and LC3-II/LC3-I ratio, whereas decreased the expression of P62 in SIMI rats. Finally, high dose VC attenuated phosphorylation of PI3K, AKT and mTOR compared with the CLP group. SIGNIFICANCE: Our results showed that high dose VC has a good protective effect on SIMI after continuous treatment, which may be mediated by inhibiting apoptosis and inflammatory, and promoting autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR pathway.

The protective effect of Macrostemonoside T from Allium macrostemon Bunge against Isoproterenol-Induced myocardial injury via the PI3K/Akt/mTOR signaling pathway.

Myocardial injury (MI) signifies a pathological aspect of cardiovascular diseases (CVDs) such as coronary artery disease, diabetic cardiomyopathy, and myocarditis. Macrostemonoside T (MST) has been isolated from Allium macrostemon Bunge (AMB), a key traditional Chinese medicine (TCM) used for treating chest stuffiness and pains. Although MST has demonstrated considerable antioxidant activity in vitro, its protective effect against MI remains unexplored. To investigate MST's effects in both in vivo and in vitro models of isoproterenol (ISO)-induced MI and elucidate its underlying molecular mechanisms. This study established an ISO-induced MI model in rats and assessed H9c2 cytotoxicity to examine MST's impact on MI. Various assays, including histopathological staining, TUNEL staining, immunohistochemical staining, DCFH-DA staining, JC-1 staining, ELISA technique, and Western blot (WB), were utilized to explore the potential molecular mechanisms of MI protection. In vivo experiments demonstrated that ISO caused myocardial fiber disorders, elevated cardiac enzyme levels, and apoptosis. However, pretreatment with MST significantly mitigated these detrimental changes. In vitro experiments revealed that MST boosted antioxidant enzyme levels and suppressed malondialdehyde (MDA) production in H9c2 cells. Concurrently, MST inhibited ISO-induced reactive oxygen species (ROS) production and mitigated the decline in mitochondrial membrane potential, thereby reducing the apoptosis rate. Moreover, pretreatment with MST elevated the expression levels of p-PI3K, p-Akt, and p-mTOR, indicating activation of the PI3K/Akt/mTOR signaling pathway and consequent protection against MI. MST attenuated ISO-induced MI in rats by impeding apoptosis through activation of the PI3K/Akt/mTOR signaling pathway. This study presents potential avenues for the development of precursor drugs for CVDs.

Purple sweet potato polysaccharide ameliorates concanavalin A-induced hepatic injury by inhibiting inflammation and oxidative stress.

BACKGROUND: Autoimmune hepatitis (AIH) is a prevalent liver disease that can potentially lead to hepatic fibrosis and cirrhosis. The prolonged administration of immunosuppressive medications carries significant risks for patients. Purple sweet potato polysaccharide (PSPP), a macromolecule stored in root tubers, exhibits anti-inflammatory, antioxidant, immune-enhancing, and intestinal flora-regulating properties. Nevertheless, investigation into the role and potential mechanisms of PSPP in AIH remains notably scarce. PURPOSE: Our aim was to explore the possible protective impacts of PSPP against concanavalin A (Con A)-induced liver injury in mice. METHODS: Polysaccharide was isolated from purple sweet potato tubers using water extraction and alcohol precipitation, followed by purification through DEAE-52 cellulose column chromatography and Sephadex G-100 column chromatography. A highly purified component was obtained, and its monosaccharide composition was characterized by high performance liquid chromatography (HPLC). Mouse and cellular models induced by Con A were set up to investigate the impacts of PSPP on hepatic histopathology, apoptosis, as well as inflammation- and oxidative stress-related proteins in response to PSPP treatment. RESULTS: The administration of PSPP significantly reduced hepatic pathological damage, suppressed elevation of ALT and AST levels, and attenuated hepatic apoptosis in Con A-exposed mice. PSPP was found to mitigate Con A-induced inflammation by suppressing the TLR4-P2X7R/NLRP3 signaling pathway in mice. Furthermore, PSPP alleviated Con A-induced oxidative stress by activating the PI3K/AKT/mTOR signaling pathway in mice. Additionally, PSPP demonstrated the ability to reduce inflammation and oxidative stress in RAW264.7 cells induced by Con A in vitro. CONCLUSION: PSPP has the potential to ameliorate hepatic inflammation via the TLR4-P2X7R/NLRP3 pathway and inhibit hepatic oxidative stress through the PI3K/AKT/mTOR pathway during the progression of Con A-induced hepatic injury. The results of this study have unveiled the potential hepatoprotective properties of purple sweet potato and its medicinal value for humans. Moreover, this study serves as a valuable reference, highlighting the potential of PSPP-1 as a drug candidate for the treatment of immune liver injury.

Probiotic-derived silver nanoparticles target mTOR/MMP-9/BCL-2/dependent AMPK activation for hepatic cancer treatment.

Recent advances in nanotechnology have offered novel ways to combat cancer. By utilizing the reducing capabilities of Lactobacillus acidophilus, silver nanoparticles (AgNPs) are synthesized. The anti-cancer properties of AgNPs have been demonstrated in previous studies against several cancer cell lines; it has been hypothesized that these compounds might inhibit AMPK/mTOR signalling and BCL-2 expression. Consequently, the current research used both in vitro and in silico approaches to study whether Lactobacillus acidophilus AgNPs could inhibit cell proliferation autophagy and promote apoptosis in HepG2 cells. The isolated strain was identified as Lactobacillus acidophilus strain RBIM based on 16 s rRNA gene analysis. Based on our research findings, it has been observed that this particular strain can generate increased quantities of AgNPs when subjected to optimal growing conditions. The presence of silanols, carboxylates, phosphonates, and siloxanes on the surface of AgNPs was confirmed using FTIR analysis. AgNPs were configured using UV-visible spectroscopy at 425 nm. In contrast, it was observed that apoptotic cells exhibited orange-coloured bodies due to cellular shrinkage and blebbing initiated by AgNP treatment, compared to non-apoptotic cells. It is worth mentioning that AgNPs exhibited remarkable selectivity in inducing cell death, specifically in HepG2 cells, unlike normal WI-38 cells. The half-maximum inhibitory concentration (IC50) values for HepG2 and WI-38 cells were 4.217 µg/ml and 154.1 µg/ml, respectively. AgNPs induce an upregulation in the synthesis of inflammation-associated cytokines, including (TNF-α and IL-33), within HepG2 cells. AgNPs co-treatment led to higher glutathione levels and activating pro-autophagic genes such as AMPK.Additionally, it resulted in the suppression of mTOR, MMP-9, BCL-2, and α-SMA gene expression. The docking experiments suggest that the binding of AgNPs to the active site of the AMPK enzyme leads to inhibiting its activity. The inhibition of AMPK ultimately results in the suppression of the mechanistic mTOR and triggers apoptosis in HepG2 cells. In conclusion, the results of our study indicate that the utilization of AgNPs may represent a viable strategy for the eradication of liver cancerous cells through the activation of apoptosis and the enhancement of immune system reactions.

Lactobacillus gasseri BNR17 Ameliorates Dexamethasone-Induced Muscle Loss in BALB/c Mice and C2C12 Myotubes.

This study aimed to investigate the effects and mechanism of Lactobacillus gasseri BNR17, a probiotic strain isolated from human breast milk, on dexamethasone-induced muscle loss in mice and cultured myotubes. BALB/c mice were intraperitoneally injected with dexamethasone, and orally administered L. gasseri BNR17 for 21 days. L. gasseri BNR17 treatment ameliorated dexamethasone-induced decline in muscle function, as evidenced by an increase in forelimb grip strength, treadmill running time, and rotarod retention time in both female and male mice. In addition, L. gasseri BNR17 treatment significantly increased the mass of the gastrocnemius and quadriceps muscles. Dual-energy X-ray absorptiometry showed a significant increase in lean body mass and a decrease in fat mass in both whole body and hind limb after treatment with L. gasseri BNR17. It was found that L. gasseri BNR17 treatment downregulated serum myostatin level and the protein degradation pathway composed of muscle-specific ubiquitin E3 ligases, MuRF1 and MAFbx, and their transcription factor FoxO3. In contrast, L. gasseri BNR17 treatment upregulated serum insulin-like growth factor-1 level and Akt-mTOR-p70S6K signaling pathway involved in protein synthesis in muscle. As a result, L. gasseri BNR17 treatment significantly increased the levels of major muscular proteins such as myosin heavy chain and myoblast determination protein 1. Consistent with in vivo results, L. gasseri BNR17 culture supernatant significantly ameliorated dexamethasone-induced C2C12 myotube atrophy in vitro. In conclusion, L. gasseri BNR17 ameliorates muscle loss by downregulating the protein degradation pathway and upregulating the protein synthesis pathway.

Celastrol enhances the viability of random-pattern skin flaps by regulating autophagy through the AMPK-mTOR-TFEB axis.

In reconstructive and plastic surgery, random-pattern skin flaps (RPSF) are often used to correct defects. However, their clinical usefulness is limited due to their susceptibility to necrosis, especially on the distal side of the RPSF. This study validates the protective effect of celastrol (CEL) on flap viability and explores in terms of underlying mechanisms of action. The viability of different groups of RPSF was evaluated by survival zone analysis, laser doppler blood flow, and histological analysis. The effects of CEL on flap angiogenesis, apoptosis, oxidative stress, and autophagy were evaluated by Western blot, immunohistochemistry, and immunofluorescence assays. Finally, its mechanistic aspects were explored by autophagy inhibitor and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. On the seventh day after surgery, the survival area size, blood supply, and microvessel count of RPSF were augmented following the administration of CEL. Additionally, CEL stimulated angiogenesis, suppressed apoptosis, and lowered oxidative stress levels immediately after elevated autophagy in ischemic regions; These effects can be reversed using the autophagy inhibitor chloroquine (CQ). Specifically, CQ has been observed to counteract the protective impact of CEL on the RPSF. Moreover, it has also been discovered that CEL triggers the AMPK-mTOR-TFEB axis activation in the area affected by ischemia. In CEL-treated skin flaps, AMPK inhibitors were demonstrated to suppress the AMPK-mTOR-TFEB axis and reduce autophagy levels. This investigation suggests that CEL benefits the survival of RPSF by augmenting angiogenesis and impeding oxidative stress and apoptosis. The results are credited to increased autophagy, made possible by the AMPK-mTOR-TFEB axis activation.

Effects of moxibustion with wheat-grain size cone at "Zusanli" (ST 36) on vascular injury and oxidative stress in high-fat diet rats through mTOR/HIF-1α/VEGF signaling pathway. / 基于mTOR/HIF-1α/VEGF信号通路探讨麦粒灸"足三里"å¯¹é«˜è„‚é¥®é£Ÿå¤§é¼ è¡€ç®¡æŸä¼¤å’Œæ°§åŒ–åº”æ¿€çš„å½±å“.

OBJECTIVES: To explore the effect mechanism of moxibustion with wheat-grain size cone at "Zusanli" (ST 36) on vascular injury and oxidative stress in hyperlipidemia through mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway. METHODS: Forty healthy male SD rats with SPF grade were randomly divided into a normal group, a model group, a moxibustion group, and an inhibitor group, with 10 rats in each one. The hyperlipidemia model was established by feeding a high-fat diet for 8 weeks in rats of the model group, the moxibustion group and the inhibitor group. The moxibustion with wheat-grain size cone was delivered at bilateral "Zusanli" (ST 36) of each rat in the moxibustion group and the inhibitor group, with 3 cones on each acupoint in each intervention, once daily for 4 weeks. In the inhibitor group, before each intervention with moxibustion, rapamycin solution was injected intraperitoneally, 2.0 mg/kg. After modeling and intervention, using ELISA, the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum of rats were determined. After intervention, with HE staining and oil red O staining adopted, the abdominal aortic morphology and peripheral lipid deposition were observed. Separately, using WST-1, TBA and micro-plate method, the superoxide dismutase (SOD) activity and the levels of malondialdehyde (MDA) and nitric oxide (NO) in the serum were detected. The protein expression of mTOR, HIF-1α and VEGF in abdominal aorta were measured by Western blot method. RESULTS: Compared with those in the normal group, the levels of TC, TG and LDL-C increased (P<0.01) and HDL-C decreased (P<0.01) in the serum of the rats in the model group, the moxibustion group and the inhibitor group after model establishment. When compared with the normal group after intervention, in the model group, the serum levels of TC, TG, LDL-C and MDA increased (P<0.01), HDL-C level, SOD activity and NO level were reduced (P<0.01); the cell structure of the abdominal arota was abnormal, the peripheral lipids deposited seriously; and the protein expression of mTOR, HIF-1α and VEGF of abdominal aorta was elevated (P<0.01, P<0.05). In comparison with the model group, the levels of TC, TG, LDL-C and MDA were reduced (P<0.01), HDL-C levels, SOD activities and NO levels elevated (P<0.01, P<0.05), as well as the protein expression of mTOR, HIF-1α and VEGF of abdominal aorta (P<0.01, P<0.05) in the moxibustion group and the inhibitor group; besides, the vascular structure was ameliorated and the lipid deposition reduced in the moxibustion group, while, the vascular structure was still abnormal and the lipid deposition declined in the inhibitor group. When compared with the inhibitor group, the serum SOD activity and NO level increased (P<0.05) and MDA decreased (P<0.05); and the protein expression of mTOR, HIF-1α and VEGF of abdominal aorta was elevated (P<0.01, P<0.05) in the moxibustion group. CONCLUSIONS: The vascular injury due to hyperlipidemia is repaired by moxibustion with wheat-grain size cone at "Zusanli" (ST 36) through ameliorating oxidative stress, which is associated potentially with the modulation of mTOR/HIF-1α/VEGF signaling pathway.

L-Glutamine Substantially Improves 5-Fluorouracil-Induced Intestinal Mucositis by Modulating Gut Microbiota and Maintaining the Integrity of the Gut Barrier in Mice.

SCOPE: This study investigates the potential of glutamine to mitigate intestinal mucositis and dysbiosis caused by the chemotherapeutic agent 5-fluorouracil (5-FU). METHODS AND RESULTS: Over twelve days, Institute of Cancer Research (ICR) mice are given low (0.5 mg kg-1) or high (2 mg kg-1) doses of L-Glutamine daily, with 5-FU (50 mg kg-1) administered between days six and nine. Mice receiving only 5-FU exhibited weight loss, diarrhea, abnormal cell growth, and colonic inflammation, correlated with decreased mucin proteins, increased endotoxins, reduced fecal short-chain fatty acids, and altered gut microbiota. Glutamine supplementation counteracted these effects by inhibiting the Toll-like receptor 4/nuclear factor kappa B (TLR4/NF-κB) pathway, modulating nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) oxidative stress proteins, and increasing mammalian target of rapamycin (mTOR) levels, thereby enhancing microbial diversity and protecting intestinal mucosa. CONCLUSIONS: These findings underscore glutamine's potential in preventing 5-FU-induced mucositis by modulating gut microbiota and inflammation pathways.

Gentisic acid prevents colorectal cancer metastasis via blocking GPR81-mediated DEPDC5 degradation.

BACKGROUND: Metastasis driven by epithelial-mesenchymal transition (EMT) remains a significant contributor to the poor prognosis of colorectal cancer (CRC), and requires more effective interventions. GPR81 signaling has been linked to tumor metastasis, while lacks an efficient specific inhibitor. PURPOSE: Our study aimed to investigate the effect and mechanism of Gentisic acid on colorectal cancer (CRC) metastasis. STUDY DESIGN: A lung metastasis mouse model induced by tail vein injection and a subcutaneous graft tumor model were used. Gentisic acid (GA) was administered by an intraperitoneal injection. HCT116 was treated with lactate to establish an in vitro model. METHODS: MC38 cells with mCherry fluorescent protein were injected into tail vein to investigate lung metastasis ability in vivo. GA was administered by intraperitoneal injection for 3 weeks. The therapeutic effect was evaluated by survival rates, histochemical analysis, RT-qPCR and live imaging. The mechanism was explored using small interfering RNA (siRNA), Western blotting, RT-qPCR and immunofluorescence. RESULTS: GA had a therapeutic effect on CRC metastasis and improved survival rates and pathological changes in dose-dependent manner. GA emerged as an GPR81 inhibitor, effectively suppressed EMT and mTOR signaling in CRC induced by lactate both in vivo and in vitro. Mechanistically, GA halted lactate-induce degradation of DEPDC5 through impeding the activation of Chaperone-mediated autophagy (CMA). CONCLUSION: CMA-mediated DEPDC5 degradation is crucial for lactate/GPR81-induced CRC metastasis, and GA may be a promising candidate for metastasis by inhibiting GPR81 signaling.

Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways.

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1 (ZO-1), PI3K, AKT and mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3K, and AKT mRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 µmol Zn/L was more effective than 100 µmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKT, and PKC mRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 µmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1, and mTOR mRNA levels. Therefore, supplemental Zn, especially 50 µmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

[Effect of Erchen Decoction on liver mitochondrial function by inhibiting mTORC1/SREBP1/CAV1 pathway in mice with high-fat diet].

This study aims to investigate the effect of Erchen Decoction(ECD) on liver mitochondrial function in mice with a high-fat diet and its possible mechanism. A total of sixty C57BL/6J mice were randomly divided into a normal group, high-fat group, ECD group, mTORC1 activator(MHY) group, ECD+MHY group, and polyene phosphatidyl choline(PPC) group, with 10 rats in each group. The normal group was given a normal diet, and the other groups were fed a high-fat diet for 20 weeks. At the 17th week, the ECD group and ECD+MHY group were given ECD(8.7 g·kg~(-1)) daily, and the PPC group was given PPC(0.18 g·kg~(-1)) daily, while the remaining groups were given normal saline(0.01 mL·g~(-1)) daily for four weeks. In the 19th week, the MHY group and ECD+MHY group were injected intraperitoneally with MHY(5 mg·kg~(-1)) every other day for two weeks. During the experiment, the general conditions of the mice were observed. The contents of triglyceride(TG) and total cholesterol(TC) in serum were measured. Morphological changes in liver tissue were examined through HE and oil red O staining. The content of adenosine triphosphate(ATP) was determined using chemiluminescence, and mitochondrial membrane potential was assessed using a fluorescence probe(JC-1). Western blot was performed to detect the expression of rapamycin target protein complex 1(mTOR1), ribosomal protein S6 kinase B1(S6K), sterol regulatory element binding protein 1(SREBP1), and caveolin 1(CAV1). RESULTS:: revealed that compared with the normal group, the mice in the high-fat group exhibited significant increases in body weight and abdominal circumference(P<0.01). Additionally, there were significant increases in TG and TC levels(P<0.01). HE and oil red O staining showed that the boundaries of hepatic lobules were unclear; hepatocytes were enlarged, round, and irregularly arranged, with obvious lipid droplet deposition and inflammatory cell infiltration. The liver ATP content and mitochondrial membrane potential decreased significantly(P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 increased significantly(P<0.01), while the expression of CAV1 decreased significantly(P<0.01). Compared with the high-fat group, the body weight and TG content of mice in the ECD group and PPC group decreased significantly(P<0.05). Improvements were observed in hepatocyte morphology, lipid deposition, and inflammatory cell infiltration. Furthermore, there were significant increases in ATP content and mitochondrial membrane potential(P<0.05 or P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly in the ECD group(P<0.01), while CAV1 expression increased significantly(P<0.01). However, the indices mentioned above did not show improvement in the MHY group. When the ECD+MHY group was compared with the MHY group, there were significant reductions in body weight and TG contents(P<0.05). The morphological changes of hepatocytes, lipid deposition, and inflammatory cell infiltration were recovered. Moreover, there were significant increases in liver ATP content and mitochondrial membrane potential(P<0.05 or P<0.05). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly(P<0.01), while CAV1 expression increased significantly(P<0.01). In conclusion, ECD can improve mitochondrial function by regulating the mTORC1/SREBP1/CAV1 pathway. This mechanism may be involved in the resolution of phlegm syndrome and the regulation of lipid metabolism.

[Total polyphenols of Cydonia oblonga inhibited proliferation and migration of renal cancer cells by PI3K/Akt/mTOR pathway].

The mechanism of total polyphenols of Cydonia oblonga Miller(TPCOM) against kidney cancer was elucidated through a combination of network pharmacology, bioinformatics, and experimental verification. The active polyphenolic compounds from C. oblonga were screened by network pharmacological techniques and kidney cancer-related targets were collected through the database. The differential gene expression analysis was performed on RNA sequencing data from tumor tissue and normal tissue of kidney cancer patients obtained from the Gene Expression Omnibus(GEO) database. The results of network pharmacology predictions and differential gene expression analysis were used to identify the core genes targeted by TPCOM in kidney cancer. Survival analysis was conducted to identify key targets that could impact patient survival, followed by Kyoto Encyclopedia of Genes and Genomes(KEGG) and Gene Ontology(GO) enrichment analyses. Cell proliferation and activity experiments(cell counting kit-8) were conducted using TPCOM at concentrations ranging from 20 to 640 µg·mL~(-1) on 786-O and Renca cells. Additionally, TPCOM at concentrations of 40, 80, and 160 µg·mL~(-1) was applied to kidney cancer cells to assess its effect on cell migration and its regulation of protein expression levels related to the protein kinase B(Akt), mammalian target of rapamycin(mTOR), and phosphoinositide 3-kinase(PI3K) signaling pathways. Network pharmacology predicted eight active polyphenolic compounds from C. oblonga. Survival analysis revealed 15 significantly differentially expressed genes in kidney cancer that were affected by TPCOM and had a significant impact on patient survival. KEGG and GO analysis results indicated that these 15 targets were primarily associated with the PI3K/Akt signaling pathway, cell migration, and proliferation. The results showed that TPCOM could inhibit the proliferation of 786-O and Renca cells, with IC_(50) values of 121.4 and 137.9 µg·mL~(-1), respectively. TPCOM was also found to inhibit the migration of these cells and suppress the PI3K/Akt/mTOR signaling pathway. TPCOM may exert its anti-kidney cancer effects by inhibiting the activation of the PI3K/Akt/mTOR signaling pathway, thereby restraining the proliferation and migration of kidney cancer cells. This study provides a foundation for the research on the anti-tumor effects of natural product C. oblonga, particularly in Xinjiang, and holds significance for further promoting its development and utilization.

Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

A Metabolomics Study of the Effects of Eleutheroside B on Glucose and Lipid Metabolism in a Zebrafish Diabetes Model.

(1) Background: Diabetes is a common metabolic disease that seriously endangers human health. In the present study, we investigated the therapeutic effects of the active ingredient Eleutheroside B (EB) from the traditional Chinese medicine Eleutheroside on diabetes mellitus in a zebrafish model. Concomitant hepatic injury was also analysed, along with the study of possible molecular mechanisms using metabolomics technology. This work should provide some theoretical references for future experimental studies. (2) Methods: A zebrafish diabetes model was constructed by soaking in a 1.75% glucose solution and feeding a high-fat diet. The intervention drug groups were metformin (100 µg∙mL-1) and EB (50, 100, and 150 µg∙mL-1) via water-soluble exposure for 30 days. Glucose, TG, TC, LDL-C, and HDL-C were evaluated in different treatment groups. GLUT4 protein expression was also evaluated in each group, and liver injury was observed by HE staining. Metabolomics techniques were used to investigate the mechanism by which EB regulates endogenous markers and metabolic pathways during the development of diabetes. (3) Results: All EB treatment groups in diabetic zebrafish showed significantly reduced body mass index (BMI) and improved blood glucose and lipid profiles. EB was found to upregulate GLUT4 protein expression and ameliorate the liver injury caused by diabetes. Metabolomics studies showed that EB causes changes in the metabolic profile of diabetic zebrafish. These were related to the regulation of purine metabolism, cytochrome P450, caffeine metabolism, arginine and proline metabolism, the mTOR signalling pathway, insulin resistance, and glycerophospholipid metabolism. (4) Conclusions: EB has a hypoglycaemic effect in diabetic zebrafish as well as significantly improving disorders of glycolipid metabolism. The mechanism of action of EB may involve regulation of the mTOR signalling pathway, purine metabolism, caffeine metabolism, and glycerophospholipid metabolism.

Investigation of the mutual crosstalk between ER stress and PI3K/AKT/mTOR signaling pathway in iron overload-induced liver injury in chicks.

Iron is an essential element for the normal functioning of living organisms, but excessive iron deposition can lead to organ damage. This study aims to investigate the interaction between the endoplasmic reticulum stress signaling pathway and the PI3K/AKT/mTOR signaling pathway in liver injury induced by iron overload in chicks. Rspectively, 150 one-day-old broilers were divided into three groups and supplemented with 50 (C), 500 (E1), and 1000 (E2) mg ferrous sulfate monohydrate/kg in the basal diet. Samples were taken after continuous feeding for 14 days. The results showed that iron overload could upregulate the levels of ALT and AST. Histopathological examination revealed bleeding in the central vein of the liver accompanied by inflammatory cell infiltration. Hoechst staining showed that the iron overload group showed significant bright blue fluorescence, and ultrastructural observations showed chromatin condensation as well as mitochondrial swelling and cristae disorganization in the iron overload group. RT-qPCR and Western blot results showed that iron overload upregulated the expression of Bax, Caspase-3, Caspase-9, GRP78, GRP94, P-PERK, ATF4, eIF2α, IRE1, and ATF6, while downregulating the expression of Bcl-2 and the PI3K/AKT/mTOR pathway. XBP-1 splicing experiment showed significant splicing of XBP-1 gene after iron overload. PCA and correlation analysis suggested a potential association between endoplasmic reticulum stress, the PI3K/AKT/mTOR signaling pathway, and liver injury in chicks. In summary, iron overload can induce cell apoptosis and liver injury by affecting endoplasmic reticulum stress and the PI3K/AKT/mTOR signaling pathway.

Melatonin counteracts cadmium-induced rat testicular toxicity via the mechanistic target rapamycin (mTOR) pathway.

The protective action of melatonin (MLT) against the harmful effects of cadmium (Cd) on testicular activity in rats has been documented previously; however, the involved molecular mechanisms have yet to be elucidated. Herein, we investigate the involvement of the mammalian target of rapamycin (mTOR) on the ability of MLT to counteract the damage induced by Cd on the rat testicular activity. Our study confirmed that Cd has harmful effects on the testes of rats and the protective action exerted by MLT. We reported, for the first time, that the addition of rapamycin (Rapa), a specific mTOR inhibitor, to animals co-treated with Cd and MLT completely abolished the beneficial effects exerted by MLT, indicating that the mTOR pathway partially modulates its helpful effects on Cd testicular toxicity. Interestingly, Rapa-alone treatment, provoking mTOR inhibition, produced altered morphological parameters, increased autophagy of germ and somatic cells, and reduced serum testosterone concentration. In addition, mTOR inhibition also reduced protein levels of markers of steroidogenesis (3ß-Hydroxysteroid dehydrogenase) and blood-testis barrier integrity (occludin and connexin 43). Finally, Rapa altered sperm parameters as well as the ability of mature spermatozoa to perform a proper acrosome reaction. Although further investigation is needed to better clarify the molecular pathway involved in MLT action, we confirm that MLT alleviating Cd effects can be used as a supplement to enhance testicular function and improve male gamete quality.

Vine tea total flavonoids activate the AMPK/mTOR pathway to amelioration hepatic steatosis in mice fed a high-fat diet.

Vine tea (Ampelopsis grossedentata), a traditional Chinese tea, is rich in flavonoids with various biological activities. Our study found that Vine tea total flavonoids (TFs) treatment reduced the body mass and blood lipid levels and improved the hepatic tissue morphology in mice fed the high-fat diet (HFD). In vivo, TF treatment activated the hepatic adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, initiated autophagy, and regulated the expression levels of proteins for lipid metabolism in those HFD-fed mice. In vitro, TF treatment dramatically reduced the lipid droplets and triacylglycerol content in HepG2 and L02 cells treated with oleic acid (OA). These were associated with the activation of the AMPK/mTOR pathway and autophagy initiation in OA-treated hepatocytes. This phenotype was abolished in the presence of 3-methyladenine, an autophagy inhibitor. Our results indicated that the TF activation of AMPK/mTOR leads to the stimulation of autophagy and a decrease in the buildup of intracellular lipids in hepatocytes, showing the potential of TF as a therapeutic agent for nonalcoholic fatty liver disease. PRACTICAL APPLICATION: Vine tea, a tea drink, has been consumed by Chinese folk for over a thousand years. The result of this study will provide evidence that vine tea total flavonoids have potential use as a functional material for the prevention and amelioration of nonalcoholic fatty liver disease.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

BACKGROUND AND AIM: Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Qi-dan-dihuang decoction ameliorates renal fibrosis in diabetic rats via p38MAPK/AKT/mTOR signaling pathway.

CONTEXT: Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. OBJECTIVE: This study reveals the mechanism by which QDD ameliorates DKD. MATERIALS AND METHODS: The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. RESULTS: A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways. DISCUSSION AND CONCLUSION: QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD.