The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

LDHA deficiency inhibits trophoblast proliferation via the PI3K/AKT/FOXO1/CyclinD1 signaling pathway in unexplained recurrent spontaneous abortion.

Dysregulated trophoblast proliferation, invasion, and apoptosis may cause several pregnancy-associated complications, such as unexplained recurrent spontaneous abortion (URSA). Recent studies have shown that metabolic abnormalities, including glycolysis inhibition, may dysregulate trophoblast function, leading to URSA. However, the underlying mechanisms remain unclear. Herein, we found that lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, was significantly reduced in the placental villus of URSA patients. The human trophoblast cell line HTR-8/SVneo was used to investigate the possible LDHA-mediated regulation of trophoblast function. LDHA knockdown in HTR-8/SVneo cells induced G0/G1 phase arrest and increased apoptosis, whereas LDHA overexpression reversed these effects. Next, RNA sequencing combined with Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the PI3K/AKT signaling pathway is potentially affected by downstream genes of LDHA. Especially, we found that LDHA knockdown decreased the phosphorylation levels of PI3K, AKT, and FOXO1, resulting in a significant downregulation of CyclinD1. In addition, treatment with an AKT inhibitor or FOXO1 inhibitor also verified that the PI3K/AKT/FOXO1 signaling pathway influenced the gene expression of CyclinD1 in trophoblast. Moreover, p-AKT expression correlated positively with LDHA expression in syncytiotrophoblasts and extravillous trophoblasts in first-trimester villus. Collectively, this study revealed a new regulatory pathway for LDHA/PI3K/AKT/FOXO1/CyclinD1 in the trophoblast cell cycle and proliferation.

The antidiabetic SGLT2 inhibitor canagliflozin reduces mitochondrial metabolism in a model of skeletal muscle insulin resistance.

AIMS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as canagliflozin (CANA) have emerged as an effective adjuvant therapy in the management of diabetes, however, past observations suggest CANA may alter skeletal muscle mass and function. The purpose of this work was to investigate the effects of CANA on skeletal muscle metabolism both with and without insulin resistance. METHODS: C2C12 myotubes were treated with CANA with or without insulin resistance. Western blot and qRT-PCR were used to assess protein and gene expression, respectively. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Mitochondrial, nuclei and lipid content were measured using fluorescent staining and microscopy. RESULTS: CANA decreased mitochondrial function and glycolytic metabolism as did insulin resistance, however, these changes occurred without significant alterations in gene expression associated with each pathway. Additionally, while insulin resistance reduced insulin-stimulated pAkt expression, CANA had no significant effect on insulin sensitivity. CONCLUSIONS: CANA appears to reduce mitochondrial and glycolytic metabolism without altering gene expression governing these pathways, suggesting a reduction in substrate may be responsible for lower metabolism.

Targeting PI3K/p-Akt/eNOS, Nrf2/HO-1, and NF-κB/p53 signaling pathways by angiotensin 1-7 protects against liver injury induced by ischemia-reperfusion in rats.

The liver is an important organ, and hepatic ischemia-reperfusion (IR) injury is a frequent pathophysiological process that can cause significant morbidity and mortality. Thus, our study aimed to investigate the effect of targeting PI3K/p-Akt/eNOS (phosphoinositide 3-kinase/phospho-protein kinase B/endothelial nitric oxide synthase), Nrf2/HO-1 (nuclear factor-erythroid 2-related factor-2/heme oxygenase-1), and NF-κB/p53 (nuclear factor-κB/tumor protein 53) signaling pathways by using angiotensin (1-7) [ang-(1-7)] against hepatic injury induced by IR. Thirty-two male rats were included in sham group, ang-(1-7)-treated group, hepatic IR group, and hepatic IR group treated with ang-(1-7). The levels of hepatic ang-(1-7), angiotensin II (Ang II), angiotensin-converting enzyme 2 (ACE2), HO-1, malondialdehyde (MDA), PI3K, and p-Akt were assessed. The expressions of eNOS and B-cell leukemia/lymphoma-2 (BCL-2) in the liver were determined. Histological assessment and immunohistochemical expression of NF-κB, p53, and Nrf2 were carried out. The levels of reduced glutathione (GSH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in serum were estimated. Results showed that administration of ang-(1-7) to hepatic IR rats led to significant amelioration of hepatic damage through a histological evaluation that was associated with significant upregulation of the expressions of PI3K/p-Akt/eNOS and Nrf2/HO-1 with downregulation of NF-κB/p53 signaling pathways. In conclusion, PI3K/p-Akt/eNOS and Nrf2/HO-1 signaling pathways are involved in the protective effects of ang-(1-7) against hepatic damage induced by IR. Therefore, ang-(1-7) can be used to prevent hepatic IR, which occurs in certain conditions such as liver transplantation, hemorrhagic shock, and severe infection.

Azilsartan prevents muscle loss and fast- to slow-twitch muscle fiber shift in natural ageing sarcopenic rats.

Sarcopenia is a musculoskeletal disease that reduces muscle mass and strength in older individuals. The study investigates the effects of azilsartan (AZL) on skeletal muscle loss in natural sarcopenic rats. Male Sprague-Dawley rats aged 4-6 months and 18-21 months were selected as young-matched control and natural-aged (sarcopenic) rats, respectively. Rats were allocated into young and old control (YC and OC) and young and old AZL treatment (YT and OT) groups, which received vehicles and AZL (8 mg/kg, orally) for 6 weeks. Rats were then sacrificed after muscle function analysis. Serum and gastrocnemius (GN) muscles were isolated for further endpoints. AZL significantly improved muscle grip strength and antioxidant levels in sarcopenic rats. AZL also restored the levels of insulin, testosterone, and muscle biomarkers such as myostatin and creatinine kinase in sarcopenic rats. Furthermore, AZL treatment improved the cellular and ultrastructure of GN muscle and prevented the shift of type II (glycolytic) myofibers to type I (oxidative) myofibers. The results showed that AZL intervention restored protein synthesis in natural sarcopenic rats by increasing p-Akt-1 and decreasing muscle RING-finger protein-1 and tumor necrosis factor alpha immunoexpressions. In conclusion, the present findings showed that AZL could be an effective intervention in treating age-related muscle impairments.

Mechanisms underlying LncRNA SNHG1 regulation of Alzheimer's disease involve DNA methylation.

Alzheimer's disease (AD) is a neurodegenerative disease associated with long non-coding RNAs and DNA methylation; however, the mechanisms underlying the role of lncRNA small nucleolar RNA host gene 1 (lncRNA SNHG1) and subsequent involvement of DNA methylation in AD development are not known. The aim of this study was to examine the regulatory mechanisms attributed to lncRNA SNHG1 gene utilizing 2 strains of senescence-accelerated mouse prone 8 (SAMP8) model of AD and compared to senescence-accelerated mouse resistant (SAMR) considered a control. Both strains of the mouse were transfected with either blank virus, psLenti-U6-SNHG1(low gene expression) virus, and psLenti-pA-SNHG1(gene overexpression) virus via a single injection into the brains for 2 weeks. At 2 weeks mice were subjected to a Morris water maze to determine any behavioral effects followed by sacrifice to extract hippocampal tissue for Western blotting to measure protein expression of p-tau, DNMT1, DNMT3A, DNMT3B, TET1, and p-Akt. No marked alterations were noted in any parameters following blank virus transfection. In SAMP8 mice, a significant decrease was noted in protein expression of DNMT1, DNMT3A, DNMT3B, and p-Akt associated with rise in p-tau and TET1. Transfection with ps-Lenti-U6-SNHG1 alone in SAMR1 mice resulted in a significant rise in DNMTs and p-Akt and a fall in p-tau and TET1. Transfection of SAMP8 with ps-Lenti-U6-SNHG1 blocked effects on overexpression noted in this mouse strain. However, knockdown of lncRNA SNHG1 yielded the opposite results as found in SAMR1 mice. In conclusion, the knockdown of lncRNA SNHG1 enhanced DNA methylation through the PI3K/Akt signaling pathway, thereby reducing the phosphorylation levels of tau in SAMP8 AD model mice with ameliorating brain damage attributed to p-tau accumulation with consequent neuroprotection.

Targeting the PI3K/pAKT/mTOR/NF-κB/FOXO3a signaling pathway for suppressing the development of hepatocellular carcinoma in rats: Role of the natural remedic Suaeda vermiculata forssk.

Liver malignancy is well recognized as a prominent health concern, with numerous treatment options available. Natural products are considered a renewable source, providing inspiring chemical moieties that could be used for cancer treatment. Suaeda vermiculata Forssk has traditionally been employed for management of hepatic conditions, including liver inflammation, and liver cirrhosis, as well as to improve general liver function. The findings of our earlier study demonstrated encouraging in vivo hepatoprotective benefits against liver injury generated by paracetamol and carbon tetrachloride. Additionally, Suaeda vermiculata Forssk exhibited cytotoxic activities in vitro against Hep-G2 cell lines and cell lines resistant to doxorubicin. The present investigation aimed to examine the potential in vivo hepatoprotective efficacy of Suaeda vermiculata Forssk extract (SVE) against hepatocellular carcinoma induced by diethylnitrosamine (DENA) in rats. The potential involvement of the PI3K/AKT/mTOR/NF-κB pathway was addressed. Sixty adult male albino rats were allocated into five groups randomly (n = 10). First group received a buffer, whereas second group received SVE only, third group received DENA only, and fourth and fifth groups received high and low doses of SVE, respectively, in the presence of DENA. Liver toxicity and tumor markers (HGFR, p-AKT, PI3K, mTOR, NF-κB, FOXO3a), apoptosis markers, and histopathological changes were analyzed. The current results demonstrated that SVE inhibited PI3K/AKT/mTOR/NF-κB pathway as well as increased expression of apoptotic parameters and FOXO3a levels, which were deteriorated by DENA treatment. Furthermore, SVE improved liver toxicity markers and histopathological changes induced by DENA administration. This study provided evidence for the conventional hepatoprotective properties attributed to SV and investigated the underlying mechanism by which its extract, SVE, could potentially serve as a novel option for hepatocellular carcinoma (HCC) treatment derived from a natural source.

Newly synthesized acridone derivatives targeting lung cancer: A toxicity and xenograft model study.

AKT is one of the overexpressed targets in nonsmall cell lung cancer (NSCLC) and plays an important role in its progression and offers an attractive target for the therapy. The PI3K/AKT/mTOR pathway is upregulated in NSCLC. Acridone is an important heterocycle compound which treats cancer through various mechanisms including AKT as a target. In the present work, the study was designed to evaluate the safety profile of three acridone derivatives (AC-2, AC-7, and AC-26) by acute and repeated dose oral toxicity. In addition to this, we also checked the pAKT overexpression and its control by these derivatives in tumor xenograft model. The results from acute and repeated dose toxicity showed these compounds to be highly safe and free from any toxicity, mortality, or significant alteration in body weight, food, and water intake in the rats. In the repeated dose toxicity, compounds showed negligible variations in a few hematological parameters at 400 mg/kg. The histopathology, biochemical, and urine parameters remained unchanged. The xenograft model study demonstrated AC-2 to be inhibiting HOP-62 induced tumor via reduction in p-AKT1 (Ser473) expression significantly. In immunofluorescence staining AC-2 treated tissue section showed 2.5 fold reduction in the expression of p-AKT1 (Ser473). Histopathology studies showed the destruction of tumor cells with increased necrosis after treatment. The study concluded that AC-2 causes cell necrosis in tumor cells via blocking the p-AKT1 expression. The findings may provide a strong basis for further clinical applications of acridone derivatives in NSCLC.

Establishing nomograms for predicting disease-free survival and overall survival in patients with breast cancer.

BACKGROUND: Prognostic factors-based nomograms have been utilised to detect the likelihood of the specific cancer events. We have focused on the roles of aldehyde dehydrogenase 1 (ALDH1) and p-AKT in predicting the prognosis of BC patients. This study was designed to establish nomograms based on the integration of aldehyde dehydrogenase 1 (ALDH1) and p-AKT in predicting the disease-free survival (DFS) and overall survival (OS) of breast cancer (BC) patients. METHODS: Demographic and clinical data were obtained from BC patients admitted to our hospital between September 2015 and August 2016. Univariate and multivariate Cox regression analyses were utilised to analyse the risk factors of recurrence and mortality. The nomograms for predicting the DFS and OS were established using the screened risk factors. Stratified analysis was performed with the cut-off value of exp (pi) of 4.0-fold in DFS and OS, respectively. RESULTS: Multivariate Cox regression analysis indicated that ALDH, p-AKT and pathological stage III were independent risk factors for the recurrence among BC patients. ALDH1, p-AKT, pathological stage III and ER-/PR-/HER2- were independent risk factors for the mortality among BC patients. The established nomograms based on these factors were effective for predicting the DFS and OS with good agreement to the calibration curve and acceptable area under the receiver operating characteristic (ROC) curve. Finally, stratified analyses showed patients with a low pi showed significant decrease in the DFS and OS compared with those of high risk. CONCLUSION: We established nomograms for predicting the DFS and OS of BC patients based on ALDH1, p-AKT and pathological stages. The ER-/PR-/HER2- may be utilised to predict the OS rather than DFS in the BC patients.

Many breast cancer patients show poor response after treatment due to recurrence and metastasis. Therefore, early prediction of the disease-free survival and overall survival is crucial to the treatment outcome and clinical decision-making. In this study, we established nomograms with the demographic and clinical data from breast cancer patients admitted to our hospital between September 2015 and August 2016. Univariate and multivariate Cox regression analyses showed that some important proteins and signalling pathways were risk factors for decreased disease-free survival and overall survival of breast cancer patients. On this basis, we established an effective nomogram for predicting the disease-free survival and overall survival of these patients based on these factors. This study offers new options in the predicting the treatment outcome of breast cancer patients.

Raltitrexed enhanced antitumor effect of anlotinib in human esophageal squamous carcinoma cells on proliferation, invasiveness, and apoptosis.

BACKGROUND: Anlotinib is a multi-targeted receptor tyrosine kinase inhibitor (TKI) which has exhibited encouraging clinical activity in advanced non-small cell lung cancer (NSCLC) and soft tissue sarcoma. Raltitrexed is well known to be effective in the treatment of colorectal cancer in China. The present study aims to investigate the combinatory antitumor effect of anlotinib and raltitrexed on human esophageal squamous carcinoma cells and further explore the molecular mechanisms in vitro. METHODS: Human esophageal squamous cell lines KYSE-30 and TE-1 were treated with anlotinib or raltitrexed, or both, then cell proliferation was measured by MTS and colony formation assay; cell migration and invasion were detected by wound-healing and transwell assays; cell apoptosis rate was studied by flow cytometry and the transcription of apoptosis-associated proteins were monitored by quantitative polymerase chain reaction (qPCR) analysis. Finally, western blot was performed to check phosphorylation of apoptotic proteins after treatment. RESULTS: Treatment with raltitrexed and anlotinib showed enhanced inhibitory effects on cell proliferation, migration and invasiveness compared with raltitrexed or anlotinib monotherapy. Meanwhile, raltitrexed combined with anlotinib strongly increased cell apoptosis percentage. Moreover, the combined treatment down-regulated mRNA level of the anti-apoptotic protein Bcl-2 and invasiveness-associated protein matrix metalloproteinases-9 (MMP-9), while up-regulated pro-apoptotic Bax and caspase-3 transcription. Western blotting showed that the combination of raltitrexed and anlotinib could inhibit the expression of phosphorylated Akt (p-Akt), Erk (p-Erk) and MMP-9. CONCLUSIONS: This study indicated that raltitrexed enhanced the antitumor effects of anlotinib on human ESCC cells by down-regulating phosphorylation of Akt and Erk, providing a novel treatment option for patients with esophageal squamous cell carcinoma (ESCC).

The effect of insulin resistance on extracellular BCAA accumulation and SLC25A44 expression in a myotube model of skeletal muscle insulin resistance.

Insulin resistance is often accompanied by elevated circulating branched-chain amino acids (BCAA). We investigated the effects of insulin resistance on the mitochondrial BCAA transporter, SLC25A44, using a myotube model of insulin resistance. Insulin sensitivity and SLC25A44 expression were assessed via Western blot. Liquid chromatography-mass spectrometry was used to evaluate extracellular BCAA media content. Insulin resistance reduced pAkt activation following insulin stimulation but did not alter SLC25A44 expression. Under select conditions, insulin resistance led to the accumulation of extracellular BCAA.

Downregulation of Pinkbar/pAKT and MMP2/MMP9 Expression in MDA-MB-231 Breast Cancer Cells as Potential Targets in Cancer Therapy by hAMSCs Secretome.

Breast cancer is one of the leading causes of cancer-related deaths among women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through Pinkbar (planar intestinal- and kidney-specific BAR domain protein), pAKT, and matrix metalloproteinases including MMP2 and MMP9 on MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using Transwell 6-well plates with a pore size of 0.4 µm. After 72 h, the hAMSCs-treated MDA-MB-231 breast cancer cells, the expression of epidermal growth factor receptor (EGFR), and c-Src (a key mediator in EGFR signaling pathway), Pinkbar, pAKT, MMP2, and MMP9 were analyzed using quantitative real time PCR and western blot methods. Based on 2D and 3D cell culture models, significant reduction of tumor cell growth and motility through downregulation of EGFR, c-Src, Pinkbar, pAKT, MMP2, and MMP9 were found in MDA-MB-231 breast cancer cells. Moreover, induction of cellular apoptosis was also reported. Our finding indicates that the hAMSCS secretome has therapeutic effects on cancer cells. To identify the details of the molecular mechanisms, more experiments will be required.

BPS-induced ovarian dysfunction: Protective actions of melatonin via modulation of SIRT-1/Nrf2/NFĸB and IR/PI3K/pAkt/GLUT-4 expressions in adult golden hamster.

Ever-increasing occurrence of plastic-manufacturing industries leads to environmental pollution that has been associated with declined human health and increased incidence of compromised reproductive health. Female subfertility/infertility is a complex phenomenon and environmental toxicants as well as lifestyle factors have a crucial role to play. Bisphenol S (BPS) was believed to be a "safer" replacement of bisphenol A (BPA) but recent data documented its neurotoxic, hepatotoxic, nephrotoxic, and reprotoxic attributes. Hence based on the scarcity of reports, we investigated molecular insights into BPS-induced ovarian dysfunction and protective actions of melatonin against it in adult golden hamsters, Mesocricetus auratus. Hamsters were administered with melatonin (3 mg/kg BW i.p. alternate days) and BPS (150 mg/kg BW orally every day) for 28 days. BPS treatment disrupted hypothalamo-pituitary-ovarian (HPO) axis as evident by reduced gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), ovarian steroids such as estradiol (E2) and progesterone (P4), thyroid hormones namely triiodothyronine (T3) and thyroxine (T4) and melatonin levels along with their respective receptors (ERα, TRα, and MT-1) thereby reducing ovarian folliculogenesis. BPS exposure also led to ovarian oxidative stress/inflammation by increasing reactive oxygen species and metabolic disturbances. However, melatonin supplementation to BPS restored ovarian folliculogenesis/steroidogenesis as indicated by increased number of growing follicles/corpora lutea and E2/P4 levels. Further, melatonin also stimulated key redox/survival markers such as silent information regulator of transcript-1 (SIRT-1), forkhead box O-1 (FOXO-1), nuclear factor E2-related factor-2 (Nrf2), and phosphoinositide 3-kinase/protein kinase B (PI3K/pAkt) expressions along with enhanced ovarian antioxidant capacity. Moreover, melatonin treatment reduced inflammatory load including ovarian nuclear factor kappa-B (NFĸB), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expressions, serum tumor necrosis factor α (TNFα), C-reactive protein (CRP) and nitrite-nitrate levels as well as upregulated ovarian insulin receptor (IR), glucose uptake transporter-4 (GLUT-4), connexin-43, and proliferating cell nuclear antigen (PCNA) expressions in ovary thereby ameliorating inflammatory and metabolic alterations due to BPS. In conclusion, we found severe deleterious impact of BPS on ovary while melatonin treatment protected ovarian physiology from these detrimental changes suggesting it to be a potential preemptive candidate against environmental toxicant-compromised female reproductive health.

Upregulation of glutamate transporter 1 by mTOR/Akt pathway in astrocyte culture during oxygen-glucose deprivation and reoxygenation.

Astrocyte-specific glutamate transporter subtype 1 (GLT-1) plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons. Although the mammalian target of rapamycin (mTOR)/protein kinase B(Akt)/nuclear factor kappa B signaling cascade is involved in the upregulation of astrocytic GLT-1 in oxygen-glucose deprivation (OGD), it is unclear whether the mTOR/Akt pathway is involved in astrocytic GLT-1 upregulation in OGD and reoxygenation (OGD/R). In this study, we found that the treatment of cultured astrocytes with rapamycin and triciribine led to the decreased astrocytes' protrusions, smaller nuclei, and an increased apoptotic rate. The inhibitors of mTOR complex 1 significantly increased the expression levels of phosphorylated Akt-Ser473 (p-Akt), phosphorylated Akt-Thr308(p-Akt), and GLT-1, while Akt-specific inhibitors blocked GLT-1 expression, suggesting that the mTOR/Akt pathway is involved in GLT-1 upregulation. We further demonstrated that astrocytes under OGD/R adapted to environmental changes through the mTOR/Akt pathway, mainly by altering cell morphology and apoptosis and upregulating the expression levels of p-Akt and GLT-1. Our results suggested that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating cell morphology, apoptosis and GLT-1 upregulation.

Therapeutic Effect of Polaprezinc on Reflux Esophagitis in the Rat Model.

BACKGROUND/AIMS: To explore the protective effects and therapeutic mechanism of Esomeprazole (PPI), polaprezinc granule (PZ), and PPI + PZ on reflux esophagitis (RE) in the rat model. METHODS: Wistar rats were randomly divided into 9 groups, which contain the control group, the acid cessation group (0.7% HCl, Q3D × 4), and the acid persistence group (0.7% HCl, Q3D × 11). PPI was administered by gavage at 8 mg·kg-1 body weight and PZ was administered by gavage at 120 mg·kg-1 body weight once a day for 15 days. The gastric cardia tissue of the feeding tube was observed under the light microscope, and the levels of interleukin-8 (IL-8) and prostaglandin E2 (PGE2) were measured by ELISA. The expression of EGFR, Akt, p-Akt, and p-mTOR was detected by Western blot. RESULTS: The ELISA results showed that the levels of IL-8 and PGE2 were significantly increased in the model group, but decreased in all groups after treatment. In the acid cessation group, PZ treatment had the most significant effect on reducing IL-8 levels and PPI + PZ treatment had the most significant effect on reducing PGE2 levels. In the acid persistence group, the PPI treatment had the most significant effect on reducing the levels of IL-8 and PGE2, and the PZ treatment could also significantly reduce their levels, close to the normal value. Western blot results showed that the expression of PI3K/Akt/mTOR pathway protein was increased in the model group, while its expression was decreased after treatment. CONCLUSIONS: Polaprezinc has a significant therapeutic effect on RE in rats, which can reduce the levels of IL-8 and PGE2 and downregulate the expression of PI3K/Akt/mTOR signal pathway protein. The efficacy of polaprezinc in the treatment of reflux esophagitis is comparable to that of PPI, and the combination of them is more effective in the reflux esophagitis treatment.

Exosome is involved in liver graft protection after remote ischemia reperfusion conditioning.

BACKGROUND: Remote ischemic perconditioning (RIPerC) has been demonstrated to protect grafts from hepatic ischemia-reperfusion injury (IRI). This study investigated the role of exosomes in RIPerC of liver grafts in rats. METHODS: Twenty-five rats (including 10 donors) were randomly divided into five groups (n = 5 each group): five rats were used as sham-operated controls (Sham), ten rats were for orthotopic liver transplantation (OLT, 5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). Liver architecture and function were evaluated. RESULTS: Compared to the OLT group, the OLT + RIPerC group exhibited significantly improved liver graft histopathology and liver function (P < 0.05). Furthermore, the number of exosomes and the level of P-Akt were increased in the OLT + RIPerC group. CONCLUSIONS: RIPerC effectively improves graft architecture and function, and this protective effect may be related to the increased number of exosomes. The upregulation of P-Akt may be involved in underlying mechanisms.

Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca2+ Signaling Pathways.

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling.

Upregulation of iNOS and phosphorylated eNOS in the implantation-induced blastocysts of mice.

Purpose: This study aimed to examine expressions of iNOS and phosphorylated eNOS (p-eNOS) in implantation-induced blastocysts. We also examined the upstream of p-eNOS. Methods: To address the protein expressions in implantation-induced blastocysts, we performed immunohistochemical analysis using a delayed implantation mouse model. Immunostaining for iNOS, p-eNOS, and p-Akt was done. To address the relationship between p-eNOS and p-Akt, activated blastocysts were treated with an Akt inhibitor, MK-2206. Results: iNOS expression was at low levels in dormant blastocysts, whereas the expression was significantly increased in the activated blastocysts. Double staining of p-eNOS and p-Akt in individual blastocysts showed colocalization of p-eNOS and p-Akt of the trophectoderm. p-eNOS and p-Akt expressions were at low levels in dormant blastocysts, whereas both of them were significantly increased in the activated blastocysts. Both dormant and activated blastocysts showed significant positive correlations between p-eNOS and p-Akt. MK-2206 treatment for activated blastocysts showed that blastocysts with lower p-Akt had significantly lower p-eNOS levels. Conclusions: iNOS and p-eNOS, Ca2+ independent NOS, are upregulated by E2 in the blastocysts during implantation activation. Furthermore, p-eNOS is upregulated in implantation-induced blastocysts downstream of p-Akt.

Branched-chain amino acids at supraphysiological but not physiological levels reduce myotube insulin sensitivity.

AIMS: Branched-chain amino acids (BCAA) are often emphasized in the diets of avid exercisers, yet population data demonstrates a correlation between circulating BCAA and insulin resistance. However, it is unclear if BCAA independently promote insulin resistance in otherwise healthy cells. The purpose of this study is to examine the effect of a BCAA mixture on muscle insulin signaling in vitro in both insulin resistant and sensitive cells. MATERIALS AND METHODS: C2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2, 2, or 20 mM (based on leucine content) for either 30 min, 1 day, or 6 days. Western blot was used to assess insulin sensitivity of cells treated with BCAA both with and without concurrent insulin resistance, and, with and without insulin stimulation. RESULTS: BCAA treatment for 1 day significantly reduced basal, but not insulin-stimulated pAkt expression. BCAA treatment for 6 days resulted in significantly reduced basal insulin signaling in healthy cells and insulin-stimulated insulin signaling in insulin resistant (but not insulin sensitive) cells. CONCLUSION: Similar to previous observations demonstrating BCAA may correlate with insulin resistance during metabolically stressed conditions, we demonstrate excessively high BCAA exposure can negatively influence basal insulin signaling, as well as insulin sensitivity in insulin resistant myotubes. However, given the intentionally high concentrations of BCAA used in this study, the extent to which these observations translate to in vivo models is unclear and warrants further investigation.

RNA-seq transcriptomic analysis of 4-octyl itaconate repressing myogenic differentiation.

BACKGROUND: The 4-octyl itaconate (OI) is a type of cell-permeable itaconate derivative. Studies have shown that with an anti-fibrotic effect in systemic sclerosis, the OI also affects osteoclast differentiation. The aim of this study was to explore the molecular mechanisms underlying the effects of OI on myoblast differentiation by RNA-seq analysis. METHODS: Myoblast proliferation, differentiation, and muscle regulatory factors were examined in C2C12 myoblasts treated with OI of various concentrations (2.5, 10, 25, 50, and 100 µmol/L). Cells were treated with the PI3K-Akt activator IGF-1 to explore the role of the PI3K-Akt pathway in OI inhibition of myogenic differentiation. The regulatory mechanisms of OI in myogenesis were further investigated by RNA-seq and subsequent gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and, gene set enrichment analysis (GSEA). RESULTS: OI of various concentrations did not show any effect during cell proliferation. During differentiation, OI inhibited the expressions of the marker of mature myotubes myosin heavy chain (MHC) and myogenin in a dose-dependent manner. OI inhibited muscle differentiation by affecting MyoD-regulated activity through inhibition of AKT1 phosphorylation. The results of the KEGG enrichment analysis and GSEA showed that OI affected multiple metabolic pathways during myogenic differentiation, including PI3K-Akt signaling, calcium signaling, and PPAR signaling. CONCLUSIONS: Our study broadens the understanding of the OI inhibition of myogenic differentiation. OI plays its functions by targeting multiple molecules and pathways, providing novel insights into the understanding of the overall effect of OI.