Eupatilin inhibits pulmonary fibrosis by activating Sestrin2/PI3K/Akt/mTOR dependent autophagy pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive chronic inflammatory disease with poor clinical outcomes and ineffective drug treatment options. Eupatilin is a major component extracted from the traditional herbal medicine Artemisia asiatica Nakai. Notably, it was demonstrated to have an anti-fibrosis effect in endometrial fibrosis, vocal fold, and hepatic fibrosis. Its role and mechanism in IPF remain unclear. METHODS: This study used the TGF-ß1-induced human embryonic lung fibroblasts (MRC-5) activation, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model. Western blot, immunofluorescence staining, quantitative real time-PCR, hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemistry were used to evaluate the effects of eupatilin on fibroblast activation, pulmonary fibrosis, and autophagy. The autophagosomes were observed with a transmission electron microscope (TEM). RNA sequencing was used to determine the signaling pathway and key regulator related to autophagy. RESULTS: Eupatilin significantly decreased the expression of Col1A1, fibronectin, α-SMA, and SQSTM1/p62. In contrast, it increased the expression of LC3B II/I and the number of autophagosomes in TGF-ß1 treated MRC-5, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model; it also alleviated bleomycin-induced lung fibrosis. The KEGG pathway mapping displayed that PI3K/Akt and Sestrin2 were associated with the enhanced fibrogenic process. Eupatilin suppressed the phosphorylation of PI3K/Akt/mTOR. Autophagy inhibitor 3-methyladenine (3-MA) and Akt activator SC-79 abrogated the anti-fibrotic effect of eupatilin. Sestrin2 expression was also downregulated in TGF-ß1 treated lung fibroblasts and lung tissues of the bleomycin-induced pulmonary fibrosis mice model. Furthermore, eupatilin promoted Sestrin2 expression, and the knockdown of Sestrin2 significantly aggravated the degree of fibrosis, increased the phosphorylation of PI3K/Akt/mTOR, and decreased autophagy. CONCLUSION: These findings indicate that eupatilin ameliorates pulmonary fibrosis through Sestrin2/PI3K/Akt/mTOR-dependent autophagy pathway.

Branched-chain amino acid supplementation does not enhance lean tissue accretion in low birth weight neonatal pigs, despite lower Sestrin2 expression in skeletal muscle.

Postnatal muscle growth is impaired in low birth weight (L) neonatal pigs. Leucine supplementation has been established as a dietary intervention to enhance muscle growth in growing animals. The aim of this study was to investigate the efficacy of supplementing L neonatal pig formulas with branched-chain amino acids (B) to enhance the rate of protein accretion. Twenty-four 3-day old pigs were divided into two groups low (L) and normal birth weight (N) based on weight at birth. Pigs were assigned to a control (C) or 1% branched-chain amino acids (B) formulas, and fed at 250 mL·kg body weight -1·d-1 for 28 d. Body weight of pigs in the L group was less than those in the N group (P < 0.01). However, fractional body weight was greater for L pigs compared with their N siblings from day 24 to 28 of feeding regardless of formula (P < 0.01). In addition, feed efficiency (P < 0.0001) and efficiently of protein accretion (P < 0.0001) were greater for L than N pigs regardless of supplementation. Pigs fed the B formula had greater plasma leucine, isoleucine, and valine concentrations compared with those fed the C formula (P < 0.05). Longissimus dorsi Sestrin2 protein expression was less for pigs in the L group compared with those in the N group (P < 0.01), but did not result in a corresponding increase in translation initiation signaling. Longissimus dorsi mRNA expression of BCAT2 was less for LB pigs compared with those in the LC group, and was intermediate for NC and NB pigs (P < 0.05). Hepatic mRNA expression of BCKDHA was greater for pigs in the L compared with those in the N groups (P < 0.05). However, plasma branched-chain keto-acid concentration was reduced for C compared with those in the B group (P < 0.05). These data suggest that branched-chain amino acid supplementation does not improve lean tissue accretion of low and normal birth weight pigs, despite a reduction in Sestrin2 expression in skeletal muscle of low birth weight pigs. The modest improvement in fractional growth rate of low birth weight pigs compared with their normal birth weight siblings was likely due to a more efficient dietary protein utilization.

Nelumbinis Stamen Ameliorates Chronic Restraint Stress-Induced Muscle Dysfunction and Fatigue in Mice by Decreasing Serum Corticosterone Levels and Activating Sestrin2.

Nelumbo nucifera Gaertn. is an important aquatic vegetable, and its dried stamen (Nelumbinis stamen, NS) is a valuable nutraceutical usually used as a herbal tea. Here, we used ultrahigh-performance liquid chromatography (UPLC)-quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography (HPLC) to chemically profile NS and quantify their main constituent flavonoids, respectively. In total, 44 components were identified, including organic acids, flavonoids, monoterpene glycosides, and fatty acids. Experimental mice were induced with fatigue by exposure to chronic restraint stress (CRS) for 8 h daily for 15 days and then treated with an aqueous extract of NS (0.5 and 1 g/kg) via gavage. NS significantly mitigated CRS-induced skeletal muscle dysfunction and fatigue in mice possibly by lowering serum corticosterone levels and restoring Sestrin2 expression in the gastrocnemius to regulate metabolism, preserve mitochondrial homeostasis, and promote antioxidant capacity. These results demonstrate that NS can be used as a nutraceutical or supplement for controlling stress-induced muscle dysfunction and fatigue.

Tanshinone IIA Regulates Keap1/Nrf2 Signal Pathway by Activating Sestrin2 to Restrain Pulmonary Fibrosis.

Tanshinone IIA (Tan-IIA) is a major component extracted from the traditional herbal medicine Danshen, which has shown antipulmonary fibrosis by suppress reactive oxygen species-mediated activation of myofibroblast. However, the exact mechanism of Tan-IIA against pulmonary fibrosis (PF) remains unclear. This work aimed to explore the underlying mechanism of the protective effects of Tan-IIA on PF. By using high-throughput RNA-Seq analysis, we have compared the genome-wide gene expression profiles and pathway enrichment of Tan-IIA-treated NIH-3T3 cells with or without transforming growth factor beta 1 (TGF-[Formula: see text]1) induction. In normal NIH-3T3 cells, Tan-IIA treatment up-regulated 181 differential expression genes (DEGs) and down-regulated 137 DEGs. In TGF-[Formula: see text]1-induced NIH-3T3 cells, Tan-IIA treatment up-regulated 709 DEGs and down-regulated 1075 DEGs, and these DEGs were enriched in extracellular matrix organization, collagen fibril organization, cell adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway and P53 signaling pathway. Moreover, there were 207 co-expressed DEGs between Tan-IIA treatment vs. the Control and TGF-[Formula: see text]1 plus Tan-IIA treatment vs. TGF-[Formula: see text]1 alone treatment, some of which were related to anti-oxidative stress. In both normal and TGF-[Formula: see text]1-induced NIH-3T3 cells, protein-protein interaction network analysis indicated that Tan-IIA can regulate the expression of several common anti-oxidant genes including Heme oxygenase 1 (Ho-1, also known as Homx1), Sestrin2 (Sesn2), GCL modifier subunit (Gclm), GCL catalytic subunit (Gclc) and Sequestosome-1 (Sqstm1). Quantitative Real-time polymerase chain reaction analysis confirmed some DEGs specifically expressing on Tan-IIA treated cells, which provided new candidates for further functional studies of Tan-IIA. In both in vitro and in vivo PF models, the protein expression of Sesn2 was significantly enhanced by Tan-IIA treatment. Overexpression and knockdown experiments showed that Sesn2 is required for Tan-IIA against TGF-[Formula: see text]1-induced myofibroblast activation by reinforcing nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated anti-oxidant response via downregulation of kelch-like ECH-associated protein 1 (Keap1). These results suggest Tan-IIA inhibits myofibroblast activation by activating Sesn2-Nrf2 signaling pathway, and provide a new insight into the essential role of Sesn2 in PF.

Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway.

PURPOSE: Scutellaria barbata D. Don (SB), mainly containing flavonoids, has been frequently used for cancer treatment. However, little research has investigated the antitumor activity of flavonoids from SB (FSB). The current study aimed to assess the antitumor effect of TFSB and elucidate the probable underlying mechanism in vivo and in vitro. STUDY DESIGN: FSB was prepared, and its chemical composition was characterized by HPLC-MS. Colorectal HCT116 cells were treated with various concentration of FSB. The viability, proliferation, apoptosis, migration, and autophagy of HCT116 cells were studied, as were further confirmed in tumor xenografts. METHODS: Cell viability and proliferation were respectively examined by MTT and EdU staining. ROS was determined with DCFH-DA, and cell apoptosis was detected using flow cytometry. Transwell and wound-healing assays were performed to evaluate cell migration. Immunofluorescence was employed to evaluate sestrin2 and ATF4 level. The protein expressions of p-AMPK, p-ULK1, p-mTOR, 4E-BP1, LC3-I/II, cleaved-caspase-3, Bax, and bcl-2 were investigated by western blot. ATF4 was overexpressed in experiments to explore the role of ATF4/sestrin2 pathway in FSB-mediated efficacy. RESULTS: FSB clearly reduced the cell viability, promoted ROS generation, and induced apoptosis in HCT116 cells by down-regulated Bcl-2, and increased cleaved-caspase-3 and Bax. Furthermore, FSB significantly inhibited migration of colorectal cells in a dose-dependent manner. Further mechanistic study indicated that FSB upregulated p-mTOR protein level, and reduced p-AMPK, p-ULK1, p-mTOR, p-4E-BP1 and LC3-I/II expression, which were major autophagy-related genes. In addition, FSB could cause downregulation of endogenous mTOR inhibitor sestrin2 and ATF4 expression. Transient overexpression of ATF4 resulted in mTOR and sestrin2 inhibition, and significantly compromised the effects of FSB on apoptosis and autophagy in HCT116 cells. CONCLUSION: Our results reveal, for the first time, that FSB exerts antitumor activity through autophagy inhibition and apoptosis induction via ATF4/sestrin2 pathway in colorectal cancer cells. Scutellaria barbata D. Don may have great potential in the application for the prevention and treatment of human colorectal cancer.

Effects of chitooligosaccharide-zinc on the ovarian function of mice with premature ovarian failure via the SESN2/NRF2 signaling pathway.

Chitooligosaccharide-zinc (COS·Zn) is a powerful anti-oxidant and anti-aging scavenger, whose anti-oxidative ability immensely exceeds vitamin C. Therefore, this study was aimed to investigate the protective effects of COS·Zn against premature ovarian failure (POF) and potential mechanisms. Female KM adult mice were divided into the following groups: a treatment group (150 mg·kg-1·d-1 COS·Zn), a treatment group (300 mg·kg-1·d-1 COS·Zn), a prevention group, two control groups and two CY/BUS groups. COS·Zn (150, 300 mg·kg-1·d-1) and COS·Zn (300 mg·kg-1·d-1) were therapeutically and preventatively administered to POF mice in the treatment and prevention studies, respectively. All the groups were administered for 21 days. Fewer primary and secondary follicles were observed in the COS·Zn-treated groups (including the treatment and prevention groups) than those of the control groups. Meanwhile, the ovarian index and the levels of FSH and LH notably increased in the treatment and prevention groups compared with those in the CY/BUS group. The levels of MVH, OCT4 and PCNA in the treatment group (300·kg-1·d-1 COS·Zn) and MVH in the prevention group remarkably increased compared with those in the CY/BUS groups. Meanwhile, the levels of P53 and P16 protein were down-regulated in the treatment and prevention groups compared with those in the CY/BUS groups. Additionally, the amounts of Sestrin2 (SESN2) and SOD2 protein were obviously higher in the treatment group (150 mg·kg-1·d-1 COS·Zn) than those in the CY/BUS groups. Similarly, the amounts of NRF2 and SESN2 protein were up-regulated in the prevention group. Besides, an increased GSH level was observed in the two treatment groups, compared with that in the CY/BUS groups, and the same trend was also present in the prevention group. Taken together, COS·Zn improves the ovarian and follicular development through regulating the SESN2/NRF2 signaling pathway. These results suggest the role of COS·Zn as a novel agent for the treatment and prevention of POF.

Pharmacokinetic Analysis of Carnosic Acid and Carnosol in Standardized Rosemary Extract and the Effect on the Disease Activity Index of DSS-Induced Colitis.

Rosemary extract (RE) is an approved food preservative in the European Union and contains dietary phytochemicals that are beneficial for gastrointestinal health. This study investigated the effects of RE on dextran sodium sulfate (DSS)-induced colitis and also determined the pharmacokinetics of dietary phytochemicals administered to mice via oral gavage. Individual components of rosemary extract were separated and identified by LC-MS/MS. The pharmacokinetics of two major diterpenes from RE, carnosic acid (CA) and carnosol (CL), administered to mice via oral gavage were determined. Then, the effect of RE pre-treatment on the disease activity index (DAI) of DSS-induced colitis in mice was investigated. The study determined that 100 mg/kg RE significantly improved DAI in DSS-induced colitis compared to negative control. Sestrin 2 protein expression, which increased with DSS exposure, was reduced with RE treatment. Intestinal barrier integrity was also shown to improve via fluorescein isothiocyanate (FITC)-dextran administration and Western blot of zonula occludens-1 (ZO-1), a tight junction protein. Rosemary extract was able to improve the DAI of DSS-induced colitis in mice at a daily dose of 100 mg/kg and showed improvement in the intestinal barrier integrity. This study suggests that RE can be an effective preventative agent against IBD.

Luteolin prevents liver from tunicamycin-induced endoplasmic reticulum stress via nuclear factor erythroid 2-related factor 2-dependent sestrin 2 induction.

Endoplasmic reticulum (ER) stress designates a cellular response to the accumulation of misfolded proteins, which is related to disease progression in the liver. Luteolin (3',4',5,7-tetrahydroxyflavone) is a phytochemical found frequently in medicinal herbs. Although luteolin has been reported to possess the therapeutic potential to prevent diverse stage of liver diseases, its role in hepatic ER stress has not been established. Thus, the present study aimed to determine the role of luteolin in tunicamycin (Tm)-induced ER stress, and to identify the relevant mechanisms involved in its hepatoprotective effects. In hepatocyte-derived cells and primary hepatocytes, luteolin significantly decreased Tm- or thapsigargin-mediated C/EBP homologous protein (CHOP) expression. In addition, luteolin reduced the activation of three canonical signaling pathways related to the unfolded protein response, and decreased mRNA levels of glucose-regulated protein 78, ER DNA J domain-containing protein 4, and asparagine synthetase. Luteolin also significantly upregulated sestrin 2 (SESN2), and luteolin-mediated CHOP inhibition was blocked in SESN2 (+/-) cells. Moreover, luteolin resulted in phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as increased nuclear Nrf2 expression. Deletion of the antioxidant response element in the human SESN2 promoter inhibited increased luciferase activation by luteolin, suggesting that Nrf2 is a critical transcription factor for luteolin-dependent SESN2 expression. In a Tm-mediated liver injury model, luteolin decreased serum alanine aminotransferase and aspartate aminotransferase activities, prevented degenerative changes and apoptosis of hepatocytes, and inhibited CHOP and glucose-regulated protein 78 expression in hepatic tissues. Therefore, luteolin may be an effective phytochemical to manage ER stress-related liver injury.

Improvement of NRF2 gene expression and antioxidant status in patients with type 2 diabetes mellitus after supplementation with omega-3 polyunsaturated fatty acids: A double-blind randomised placebo-controlled clinical trial.

BACKGROUND: Nrf2 is a transcription factor that induces the expression of several proteins with antioxidant properties such as sestrin2 (Sesn2) and is therefore considered as the major regulator of anti-oxidative defence system. OBJECTIVES: The aim of this research was to study the effect of supplementation with n-3 PUFAs on the antioxidant status and the gene expression of Nrf2 and Sestrin2 (Sesn2) in patients with type 2 diabetes mellitus (T2DM). PARTICIPANTS: Sixty patients with T2DM were enrolled in a placebo-controlled, double-blind, randomised clinical trial. INTERVENTION AND DESIGN: The participants were randomly allocated to two intervention groups receiving either n-3 PUFAs (2,700 mg/day) (n = 30) or placebo soft gels containing 900 mg of edible paraffin (n = 30). The main outcome measures were the expression of Sesn2 and Nrf2 genes which were assessed in peripheral blood mononuclear cells (PBMCs) after RNA extraction and cDNA synthesis by real-time PCR. Total antioxidant status in plasma samples was also measured based on the ferric reducing ability of plasma. RESULTS: NRF2 gene expression was significantly increased in n-3 PUFA-supplemented subjects, compared with the placebo group. Plasma total antioxidant status was also significantly augmented in n-3 PUFA-supplemented subjects. SESN2 gene expression was not significantly affected by n-3 PUFA supplementation although a slight up-regulation was observed. CONCLUSION: Supplementation with n-3 PUFAs enhanced NRF2 gene expression and improved overall antioxidant capacity and thus might be considered beneficial in the amelioration of oxidative stress and prevention of T2DM complications. TRIAL REGISTRATION: IRCT20150926024198N4.

Effect of exercise and butyrate supplementation on microbiota composition and lipid metabolism.

The composition and activity of the gut microbiota depend on the host genome, nutrition, and lifestyle. Exercise and sodium butyrate (NaB) exert metabolic benefits in both mice and humans. However, the underlying mechanisms have not been fully elucidated. This study aimed to examine the effect of exercise training and dietary supplementation of butyrate on the composition of gut microbiota and whether the altered gut microbiota can stimulate differential production of short-chain fatty acids (SCFAs), which promote the expression of SESN2 and CRTC2 to improve metabolic health and protect against obesity. C57BL/6J mice were used to study the effect of exercise and high-fat diet (HFD) with or without NaB on gut microbiota. Bacterial communities were assayed in fecal samples using pyrosequencing of 16S rRNA gene amplicons. Western blot was performed using relevant antibodies to detect the protein expressions in liver and HepG2 cell extracts. Exercise and butyrate administration significantly reversed metabolic dysfunctions induced by HFD (P < 0.05). The number of Firmicutes and the proportion of Firmicutes to Bacteroidetes order were predominant in all HFD groups (P = 0.001). Exercise and butyrate supplementation significantly inhibited the relative abundance of lipopolysaccharide-producing phyla (P = 0.001). SESN2 and CRTC2 expression in the liver of mice were significantly increased after exercise (P < 0.05) and/or supplementation of butyrate (P < 0.05). Exercise enhances butyrate-producing fecal bacteria and increases butyrate production and consequently improves lipid metabolism through the butyrate-SESN2/CRTC2 pathway. Excess butyrate may reduce the proportion of probiotics and reverse the metabolic effects.

Sestrin2 Negatively Regulates Casein Synthesis through the SH3BP4-mTORC1 Pathway in Response to AA Depletion or Supplementation in Cow Mammary Epithelial Cells.

Sestrin2 (SESN2) negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) pathway and casein synthesis in response to amino acid (AA) depletion in cow mammary epithelial cells (CMECs); however, the underlying mechanism is unclear. In the current study, the regulation of SESN2 on AA-mediated ß-casein (CSN2) synthesis in CMECs and its mechanism were investigated. Overexpression and silencing of SESN2 demonstrated that SESN2 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway. Co-immunoprecipitation analysis showed that SESN2 interacted with SH3 domain-binding protein 4 (SH3BP4). Overexpression and silencing of SH3BP4 demonstrated that SH3BP4 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway and that SESN2 negatively regulated expression of CSN2 and mTORC1 pathway through the SH3BP4 in the presence and absence of AA. The absence or presence of AA demonstrated that AA negatively regulated expression and nuclear localization of activating transcription factor 4 (ATF4). Overexpression and silencing of ATF4 demonstrated that AA negatively regulated SESN2 expression through ATF4. Together, these results indicate that SESN2 negatively regulates the mTORC1 pathway and subsequent CSN2 synthesis through the SH3BP4 in response to AA absence or presence in CMECs.

Arsenic trioxide-mediated suppression of miR-182-5p is associated with potent anti-oxidant effects through up-regulation of SESN2.

Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 (SESN2) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3'- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

SESN2/sestrin 2 induction-mediated autophagy and inhibitory effect of isorhapontigenin (ISO) on human bladder cancers.

Isorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 µM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.