Cell-cell crosstalk between fat cells and immune cells.

Obesity is a metabolic disorder with pandemic-like implications, lacking viable pharmaceutical treatments currently. Thermogenic adipose tissues, including brown and beige adipose tissues, play an essential role in regulating systemic energy homeostasis and have emerged as appealing therapeutic targets for the treatment of obesity and obesity-related diseases. The function of adipocytes is subject to the complex regulation by a cellular network of immune signaling pathways in response to environmental signals. However, the specific regulatory roles of immune cells in thermogenesis and relevant involving mechanisms are still not well understood. Here, we concentrate on our present knowledge of the interaction between thermogenic adipocytes and immune cells, present an overview of cellular and molecular mechanisms underlying immunometabolism in adipose tissues. We discuss cytokines, especially interleukins, which are originated from widely variable sources and their impacts on the development and function of thermogenic adipocytes. Moreover, we summarize the neuro-immune regulation in heat production and expand a new mode of intercellular communication mediated by mitochondrial transfer. The crosstalk between immune cells and adipocytes achieves adipose tissue homeostasis and systemic energy balance. A deep understanding of this intricate interaction would provide evidence on improving thermogenic efficiency by remodeling the immune microenvironment. Interventions based on these factors show high potential to prevent adverse metabolic outcomes in obese patients.

The synthesis and evaluation of novel ALK inhibitors containing the sulfoxide structure.

With ceritinib as the lead, a series of novel compounds containing the sulfoxide structure were synthesized and evaluated as anaplastic lymphoma kinase inhibitors. Among them, compounds 18a-d exhibited excellent anti-proliferation activities on H2228 EML4-ALK cancer cell lines with 14-28 nM of the IC50 values. In xenograft mouse models, 18a-d inhibited tumor growth with an excellent inhibitory rate of 75.0% to 86.0% at the dosage of 20 mg kg-1 as compared to 72.0% of the reference ceritinib. Using 18d as a representative, which exhibited the best in vivo results, we carried out mechanistic studies such as anti-colony formation, induced tumor cell apoptosis, ALK kinase protein phosphorylation in H2228 tumor cells, and molecular docking. All these results indicate that compound 18d is a good anti-tumor lead compound and worthy of further study.

Discovery of new cyclopropane sulfonamide derivatives as EGFR inhibitors to overcome C797S-mediated resistance and EGFR double mutation.

The C797S mutation of EGFR leads to Osimertinib resistance by blocking the covalent binding of Cys797. To develop new agents that can overcome EGFR mutation resistance, thirty seven new cyclopropane sulfonamide derivatives were synthesized and evaluated as EGFRL858R/T790M/C797S or EGFRDel19/T790M/C797S inhibitors by structure-based screening. Most of the synthesized compounds exhibit good to excellent anti proliferation activity against to BaF3-EGFR L858R/T790M/C797S and BaF3-C797S/Del19/T790M cancer cell lines. Representative compounds 8l showed inhibitory activity against the two cancer cell lines with the IC50 values of 0.0012 and 0.0013 µM, respectively. Another compound 8h, exhibited slightly lower activity (0.0042 and 0.0034 µM of the IC50 values) to both of the two tri-mutation cell lines, but excellent activities against H1975 and PC9 cells with IC50 values of 13 and 19 nM, respectively. Considering the acquired drug resistance of tumors is a gradual process, we chose 8h for further in vivo and mechanism study. 8h was demonstrated significantly inhibited tumor growth with 72.1 % of the TGI in the BaF3/EGFR-TM xenograft tumor model and 83.5 % in the H1975-DM xenograft tumor model. Compound 8h was confirmed to be safe with no significant side effects as showed by the results of in vitro assay of human normal cells and the sections of animals major organs. Mechanism studies showed that in addition to inhibiting EGFR mutations, 8h can also target the tumor microenvironment and induce tumor cell apoptosis. All these results indicate that 8h deserves further investigation as an EGFR inhibitor to overcome C797S-mediated resistance.

The new N2, N4-diphenylpyridine-2,4-diamine deuterated derivatives as EGFR inhibitors to overcome C797S-mediated resistance.

A series of new deuterated and non-deuterated N2, N4-diphenylpyridine - 2,4-diamine derivatives were synthesized and evaluated as EGFR C797S-mediated resistance inhibitors. Most of these compounds exhibited potent antiproliferative activity against Baf3-EGFR L858R/T790M/C797S and Baf3-EGFR Del19/T790M/C797S cancel cell lines, with IC50 values in the nanomolar concentration range. Among them, compound 14l represented the most active compound with IC50 values of 8-11 nM. Interestingly, metabolic stability assay with rat liver microsomes indicated that the half-life of the deuterated derivative 14o was significantly increased compared to that of 14l. In xenograft mice models, 14o inhibited tumor growth with excellent inhibitory rate of 75.1 % at the dosage of 40 mg/kg, comparing 73.2 % of the TGI with its non-deuterated compound 14l, at a dosage of 80 mg/kg. Mechanism studies revealed that 14o was a potent EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S kinase inhibitor, which could downregulate the protein phosphorylation of EGFR and m-TOR signaling pathways, arrest cell cycle at G2/M phase by affecting the expression of CDC25C, and promote cell apoptosis by regulating the expression of cleaved caspase-3. In summary, 14o could serve as a promising deuterated compound for the development of highly efficient anticancer agents.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis.

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Growth and Hepatopancreas Health of Juvenile Chinese Mitten Crab (Eriocheir sinensis) Fed Different Levels of Black Soldier Fly (Hermetia illucens) Larvae Meal for Fish Meal Replacement.

A 56-day feeding trial assessed the effects of black soldier fly larvae meal (BSFLM) on the growth performance and hepatopancreas health of juvenile Eriocheir sinensis. Six isoproteic and isolipidic diets with 0% (FM), 10% (BSFLM10), 20% (BSFLM20), 30% (BSFLM30), 40% (BSFLM40), or 50% (BSFLM50) replacement of fish meal by BSFLM were formulated. Compared to FM, replacing 10%-40% of fish meal with BSFLM did not significantly affect the weight gain rate (WGR) or specific growth rate (SGR), while BSFLM50 significantly decreased the WGR and SGR. Crabs fed BSFLM50 had significantly lower T-AOC activity than those fed other diets, and crabs fed BSFLM30, BSFLM40, or BSFLM50 had significantly lower activities of antioxidant enzymes (SOD and GSH-Px) in the hepatopancreas than those fed FM or BSFLM10. Compared to FM, BSFLM10, BSFLM20, and BSFLM30 did not affect the relative expression of genes related to the nonspecific immunity, while BSFLM40 and BSFLM50 upregulated the relative expression of these genes. Furthermore, histological analysis showed that the hepatopancreas was deformed in the BSFLM50 group, with widened lumens and loss of basal membrane integrity. In summary, BSFLM replacing 50% of fish meal reduced growth and structural damage to the hepatopancreas. An immune response was activated when the replacement level was over 30%. Therefore, the replacement level of dietary fish meal by BSFLM is recommended to be not more than 30% of the juvenile E. sinensis feed.

A novel c-Met/TRK inhibitor 1D228 efficiently inhibits tumor growth by targeting angiogenesis and tumor cell proliferation.

Multiple tumors are synergistically promoted by c-Met and TRK, and blocking their cross-signalling pathway may give better effects. In this study, we developed a tyrosine kinase inhibitor 1D228, which exhibited excellent anti-tumor activity by targeting c-Met and TRK. Models in vitro, 1D228 showed a significant better inhibition on cancer cell proliferation and migration than the positive drug Tepotinib. Models in vivo, 1D228 showed robust anti-tumor effect on gastric and liver tumor growth with 94.8% and 93.4% of the TGI, respectively, comparing 67.61% and 63.9% of Tepotinib. Importantly, compared with the combination of Larotrectinib and Tepotinib, 1D228 monotherapy in MKN45 xenograft tumor models showed stronger antitumor activity and lower toxicity. Mechanistic studies showed that 1D228 can largely inhibit the phosphorylation of TRKB and c-Met. Interestingly, both kinases, TRKs and c-Met, have been found to be co-expressed at high levels in patients with gastric cancer through IHC. Furthermore, bioinformatics analysis has revealed that both genes are abnormally co-expressed in multiple types of cancer. Cell cycle analysis found that 1D228 induced G0/G1 arrest by inhibiting cyclin D1. Additionally, vascular endothelial cells also showed a pronounced response to 1D228 due to its expression of TRKB and c-Met. 1D228 suppressed the migration and tube formation of endothelial cells, which are the key functions of tumor angiogenesis. Taken together, compound 1D228 may be a promising candidate for the next generation of c-Met and TRK inhibitors for cancer treatment, and offers a novel potential treatment strategy for cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them.

Treatment of obesity-related diabetes: significance of thermogenic adipose tissue and targetable receptors.

Diabetes mellitus is mainly classified into four types according to its pathogenesis, of which type 2 diabetes mellitus (T2DM) has the highest incidence rate and is most relevant to obesity. It is characterized by high blood glucose, which is primarily due to insulin resistance in tissues that are responsible for glucose homeostasis (such as the liver, skeletal muscle, and white adipose tissue (WAT)) combined with insufficiency of insulin secretion from pancreatic ß-cells. Treatment of diabetes, especially treatment of diabetic complications (such as diabetic nephropathy), remains problematic. Obesity is one of the main causes of insulin resistance, which, however, could potentially be treated by activating thermogenic adipose tissues, like brown and beige adipose tissues, because they convert energy into heat through non-shivering thermogenesis and contribute to metabolic homeostasis. In this review, we summarize the function of certain anti-diabetic medications with known thermogenic mechanisms and focus on various receptor signaling pathways, such as previously well-known and recently discovered ones that are involved in adipose tissue-mediated thermogenesis and could be potentially targeted to combat obesity and its associated diabetes, for a better understanding of the molecular mechanisms of non-shivering thermogenesis and the development of novel therapeutic interventions for obesity-related diabetes and potentially diabetic complications.

Synthesis and evaluation of sulfonamide derivatives targeting EGFR790M/L858R mutations and ALK rearrangement as anticancer agents.

Fourteen new compounds bearing sulfonamide groups that target EGFRT790M/L858R mutations and ALK rearrangement were synthesized and evaluated as dual-target tumor inhibitors. The study on the anti-proliferation activity on cancer cells showed that the sulfonamide derivative with pyrimidine nucleus had much better activities compared with those with quinazoline nucleus. Among them, compound 19e exhibited excellent activity against H1975 cancer cell lines (EGFRT790M/L858R high express) and H2228 cells (ALK rearrangement) with the IC50 values of 0.0215 µM and 0.011 µM, respectively. The ALK and EGFR kinase inhibition assays also provided similar results. Genotype selectivity of EGFR on kinase and cell level, cytotoxicity towards human normal cell lines and cell morphology assay implied that 19e had acceptable selectivity and low toxicity. In addition, the inhibitory activity of 19e on H1975 and H2228 cells cloning and its apoptosis-inducing effect on the two cell lines were studied, and its inhibitory effect on the invasion and migration of tumor cells were also investigated. All the results show that 19e is worthy of further study.

Comprehensive transcriptional and metabolomic analysis reveals the neuroprotective mechanism of dietary gamma-aminobutyric acid response to hypoxic stress in the Chinese mitten crab (Eriocheir sinensis).

Hypoxia is one of the serious stress challenges that aquatic animals face throughout their life. Our previous study found that hypoxia stress could induce neural excitotoxicity and neuronal apoptosis in Eriocheir sinensis, and observed that gamma-aminobutyric acid (GABA) has a positive neuroprotective effect on juvenile crabs under hypoxia. To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E. sinensis exposed to hypoxia stress, an 8-week feeding trial and acute hypoxia challenge were performed. Subsequently, we performed a comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia of juvenile crabs. Differential genes and differential metabolites were co-annotated to 11 KEGG pathways, and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched. In the sphingolipid signaling pathway, GABA treatment significantly increased long-chain ceramide content in thoracic ganglia, which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis. Moreover, in the arachidonic acid metabolism pathway, GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection. Furthermore, the decrease of glucose and lactate levels in the hemolymph suggests the positive role of GABA in metabolic regulation. This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E. sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals.

Novel Dual-Target Kinase Inhibitors of EGFR and ALK Were Designed, Synthesized, and Induced Cell Apoptosis in Non-Small Cell Lung Cancer.

ALK-positive NSCLC coexisting with EGFR mutations is a frequently occurring clinical phenomenon. Targeting ALK and EGFR simultaneously may be an effective way to treat these cancer patients. In this study, we designed and synthesized ten new dual-target EGFR/ALK inhibitors. Among them, the optimal compound 9j exhibited good activity with IC50 values of 0.07829 ± 0.03 µM and 0.08183 ± 0.02 µM against H1975 (EGFR T790M/L858R) and H2228 (EML4-ALK) cells, respectively. Immunofluorescence assays indicated that the compound could simultaneously inhibit the expression of phosphorylated EGFR and ALK proteins. A kinase assay demonstrated that compound 9j could inhibit both EGFR and ALK kinases; thus, exerting an antitumor effect. Additionally, compound 9j induced apoptosis in a dose-dependent manner and inhibited the invasion and migration of tumor cells. All of these results indicate that 9j is worthy of further study.

Dietary Methionine Level Impacts the Growth, Nutrient Metabolism, Antioxidant Capacity and Immunity of the Chinese Mitten Crab (Eriocheir sinensis) under Chronic Heat Stress.

This study examined whether diets with high dietary methionine levels could alleviate chronic heat stress in Chinese mitten crab Eriocheir sinensis. Crabs were fed three dietary methionine levels of 0.49%, 1.29% and 2.09% for six weeks. The analyzed methionine concentration of diets was 0.48%, 1.05% and 1.72%, respectively. Crabs were fed three different supplemental concentrations of dietary methionine at 24 °C and 30 °C, respectively. The trial was divided into six groups with five replicates in each group, and 40 juvenile crabs (initial average weight 0.71 ± 0.01 g) in each replicate. During the trial, crabs were fed twice daily (the diet of 4% of the body weight was delivered daily). The effects of dietary methionine level on nutrient metabolism, antioxidant capacity, apoptosis factors and immunity were evaluated at a normal water temperature of 24 °C and high temperature of 30 °C. Feed conversion ratio decreased under chronic heat stress. Chronic heat stress increased weight gain, specific growth rate, molting frequency, and protein efficiency ratio. The survival of crabs decreased under chronic heat stress, whereas a high level of dietary methionine significantly improved survival. Chronic heat stress induced lipid accumulation and protein content reduction. The high-methionine diet decreased lipid in the body and hepatopancreas, but increased protein in the body, muscle and hepatopancreas under chronic heat stress. Simultaneously, the high dietary methionine levels mitigated oxidative stress by reducing lipid peroxidation, restoring the antioxidant enzyme system, decreasing apoptosis and activating immune function under chronic heat stress. This study suggests that supplementing 1.72% dietary methionine could alleviate the adverse effects of a high water temperature in E. sinensis farming.

Discovery of a potent EGFR and ALK dual mutation inhibitor containing N-(3-((4-((2-(cyclopropylsulfinyl)phenyl)amino)pyrimidin-2-yl)amino) phenyl)acrylamide scaffold.

A series of EGFR and ALK dual inhibitors containing sulfoxide and cyclopropyl groups were designed and synthesized. The lead compound 8a showed a significant activity against EGFR and ALK in both the enzymatic and cellular assays. The study of anti-tumor mechanism indicated that 8a could effectively block the phosphorylation of EGFR and ALK proteins, so as to effectively inhibiting the proliferation and inducing apoptosis of H1975 tumor cells, blocking the cell cycle and reducing the mitochondrial membrane potential inhibited the migration of H1975 cells. In vivo studies, compounds 8a and 8d can significantly subside the tumor tissue of nude mice without obvious toxicity.

Design, Synthesis, and Evaluation of New Mesenchymal-Epithelial Transition Factor (c-Met) Kinase Inhibitors with Dual Chiral Centers.

A series of tepotinib derivatives with two chiral centers was designed, synthesized, and evaluated as anticancer agents. The optimal compound (R, S)-12a strongly exhibited antiproliferative activity against MHCC97H cell lines with an IC50 value of 0.002 µM, compared to tepotinib (IC50 = 0.013 µM). Mechanistic studies revealed that compound (R, S)-12a significantly inhibited c-Met activation, as well as the downstream AKT signaling pathway, and suppressed wound closure. Moreover, compound (R, S)-12a induced cellular apoptosis and cell cycle arrest at the G1 phase in a dose-dependent fashion.

Combined effects of polystyrene microplastics and copper on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia (Oreochromis niloticus).

Microplastics (MPs) coexist with other pollutants (such as heavy metals) in water, adversely impacting aquatic organisms, which might cause unpredictable ecological risks. This study aims to evaluate the effect of copper (Cu2+) and polystyrene microplastics (PS-MPs) on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia. Cu2+ and PS-MPs co-exposure enhanced Cu2+ bioaccumulation in the liver of fish compared with Cu2+-alone exposure. Fish exposed to PS-MPs and Cu2+ displayed histopathologic alterations in the liver, intestine and gill. Exposure at low concentrations of Cu2+ in the C0 and CP0 groups can improve antioxidant capacity and immune response, while oxidative damage and inflammation existed in the high concentration of Cu2+ groups. Intestinal microbiota results showed that the diversity and structure were changed by Cu2+ and PS-MPs exposure, and harmful bacterium even increased at high concentration of Cu2+ and PS-MPs exposure groups. All in all, PS-MPs aggravate the accumulation of Cu2+ and lead to perturbations in biological systems of Nile tilapia.

Acute exposure to microcystin-LR induces hepatopancreas toxicity in the Chinese mitten crab (Eriocheir sinensis).

The Chinese mitten crab is an important economic species in the Chinese aquaculture industry due to its rich nutritional value and distinct flavor. The hepatopancreas is a popular edible part of the Chinese mitten crab, and therefore, hepatopancreatic health directly determines its quality. However, a large-scale outbreak of hepatopancreatic necrosis syndrome ("Shuibiezi" disease in Chinese), which is caused by abiotic agents correlated with cyanobacteria bloom outbreaks, adversely affects the Chinese mitten crab breeding industry. Cyanobacterial blooms that occur in high-density farming ponds can produce microcystin-LR (MC-LR), which is hepatotoxic in fish and mammals. Hepatopancreas toxicity of MC-LR (0, 25, 50 and 75 µg/kg) was investigated after 48 h of exposure. The MC-LR can cause hepatopancreatic injury by inducing hepatopancreatic structural damage, subcellular structural changes, and cell apoptosis, followed by enhanced lipid peroxidase, reactive oxygen species, and apoptosis-related enzyme (Caspase 3, 8, and 9) activities. These in turn promote gene and protein expression of apoptosis-associated proteases (Caspase 3, 7, and 8, Bcl-2, and Bax), and alter antioxidant system responses (superoxide dismutase, glutathione S-transferase, glutathione peroxidase, glutathione reductase activities, and glutathione content). The present study is the first report on MC-LR hepatotoxicity in the Chinese mitten crab and confirms hepatopancreas toxicity, providing a theoretical basis for enhancing MCs resistance and developing preventive and curative measures against hepatopancreatic disease in the Chinese mitten crab breeding industry.

Protective effects of dietary arginine against oxidative damage and hepatopancreas immune responses induced by T-2 toxin in Chinese mitten crab (Eriocheir sinensis).

T-2 toxin is a secondary metabolite produced by Fusarium spp. that is a major cereal and animal feed contaminant. T-2 toxin has numerous adverse effects on animals, including hepatotoxicity. Arginine (Arg) is closely associated with the regulation of immune responses and antioxidant activity in tissues. The objective of the present study was to evaluate the protective effects of dietary Arg against oxidative damage and immune responses of the hepatopancreas induced by T-2 toxin in Chinese mitten crab. According to the results, 3.17% Arg in the diet decreased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity in the haemolymph significantly, when compared with the levels of activity in the T-2 toxin group. Arg supplementation also increased superoxide dismutase and glutathione peroxidase activity, while decreasing malondialdehyde concentrations in the hepatopancreas, when compared with the levels in the T-2 toxin group. In addition, 3.17% Arg in the diet increased acid phosphatase and alkaline phosphatase activity in the hepatopancreas, as well as albumin concentrations in the haemolymph, when compared with the T-2 toxin group. Dietary Arg also regulated the expression of antioxidant enzyme-related genes (mitochondrial manganese superoxide dismutase, cytosolic manganese superoxide dismutase, and catalase) and immune related genes (prophenoloxidase, NF-κB-like transcription factor Relish, and lipopolysaccharide-induced TNF-α factor) to alleviate the damage associated with the T-2 toxin. Furthermore, Arg ameliorated damage to the hepatopancreas microstructure in the crabs. The results of the present study indicate that dietary Arg could enhance the antioxidant and immune capacity of Chinese mitten crab against oxidative damage and immune injury to the hepatopancreas induced by T-2 toxin.

Dietary reduced glutathione supplementation can improve growth, antioxidant capacity, and immunity on Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis is an important aquaculture species in China, and its yield and quality are threatened by oxidative stress caused by deteriorating water conditions. Reduced glutathione (GSH) is an endogenous antioxidant, but whether dietary GSH can increase the resistance of E. sinensis to environmental stress remains unclear. Therefore, in this study, crabs were fed with dietary GSH (0, 300, 600, 900, and 1200 mg/kg diet weight) for up to 10 weeks to determine the effects of different dietary GSH concentrations on growth, antioxidant capacity, and immunity of E. sinensis. The results showed that the weight gain rate and survival rate increased significantly as dietary GSH levels increased from 0 to 900 mg/kg, but decreased at 1200 mg/kg. Compared with the control group, the diet supplemented with 900 mg/kg GSH not only increased the concentration of GSH in the haemolymph and hepatopancreas, but also enhanced the activity of glutathione peroxidase (GSH-Px) (p < 0.05). Diets supplemented with 600 or 900 mg/kg GSH significantly increased the enzymes activities of superoxide dismutase (SOD), lysozyme (LZM), alkaline phosphatase, and acid phosphatase, and significantly decreased the content of malondialdehyde. To understand the changes in the activity of these enzymes further, the expression of related genes was detected. Diets supplemented with 600 or 900 mg/kg GSH significantly upregulated the genes expressions of cytosolic manganese SOD, mitochondrial manganese SOD, copper, zinc-SOD, GSH-Px, LZM, and prophenoloxidase activating factor, and significantly down regulated the expression of Toll-like receptor 1, Toll-like receptor 2, Dorsal, and the myeloid differentiation factor 88. However, a diet supplemented with 1200 mg/kg GSH decreased those positive indicators. Overall, our results demonstrated that an appropriate diet supplemented with 600 or 900 mg/kg GSH enhances antioxidant capacity and immunity, which will enhance the general health of E. sinensis.

Dietary glutathione supplementation enhances antioxidant activity and protects against lipopolysaccharide-induced acute hepatopancreatic injury and cell apoptosis in Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis (E. sinensis) is an important aquaculture species in China. However, deteriorating water environments lead to oxidative stress in these crabs, which subsequently reduces their quality and yield. Glutathione (GSH) is an endogenous antioxidant that is used to mitigate oxidative stress. However, whether dietary GSH can enhance the resistance of E. sinensis to oxidative stress remains unclear. Herein, crabs were fed dietary GSH (the basal diet was supplemented with 0, 300, 600, 900, and 1200 mg/kg diet weight of GSH) for up to 3 weeks and, then, challenged with lipopolysaccharide (LPS; 400 µg/kg body weight). After 6 h, their hepatopancreas were sampled. Diet supplementation with 600 and 900 mg/kg diet weight GSH not only increased the content of GSH in the hepatopancreas, but also enhanced the activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) (P < 0.05), compared to that in control crabs challenged with LPS alone. Diet supplementation with 600 or 900 mg/kg GSH also significantly increased the enzyme activities of GSH reductase and γ-glutamyl cysteine synthetase (γ-GCS) in LPS-treated crabs. Haematoxylin-eosin (HE) staining, electron microscopy, and flow cytometry were used to examine the structure and subcellular structure of and apoptosis in the hepatopancreas. The histopathology and sub-microstructure analysis results also showed that diet supplementation with 600 or 900 mg/kg GSH significantly alleviated damage in crabs challenged with LPS and decreased reactive oxygen species (ROS) levels and cell apoptosis ratios in the hepatopancreas, compared to the LPS-treated crabs. To further understand the effect of dietary GSH on LPS-induced apoptosis, the activities and gene or protein expressions of apoptosis-related factors were evaluated. As a result, diet supplementation with 600 or 900 mg/kg GSH significantly decreased the activities of caspases-3, -8, and -9 and inhibited the relative expression of caspase-3 and -8 but increased the expression of B-cell lymphoma-2 (bcl-2) and B-cell lymphoma-2-associated X inhibitor (bax inhibitor) in crabs challenged with LPS. This treatment further significantly downregulated the relative protein levels of caspase-3, -8, -9 and Bax and upregulated those of Bcl-2 in crabs challenged with LPS. However, treatment with 1200 mg/kg GSH caused the opposite effects. Overall, our results reveal that appropriate diets supplemented with 600 or 900 mg/kg GSH could enhance the antioxidant capacity and anti-apoptotic mechanisms in crabs after LPS injection, thereby providing a theoretical basis for the application of dietary GSH in E. sinensis.