mRNA Expression Of Anti-inflammatory Cytokines Research Articles

Effects of the Replacement of Dietary Fishmeal by the Blend of Tenebrio molitor Meal, Chlorella Meal, Clostridium Autoethanogenum Protein, and Cottonseed Protein Concentrate on Growth, Protein Utilization, and Intestinal Health of Gibel Carp (Carassius gibelio, CAS Ⅴ)

The trial was conducted to investigate the effects of the replacement of dietary fishmeal (FM) by the blend of Tenebrio molitor meal (TMM), Chlorella meal (CM), Clostridium autoethanogenum protein (CAP), cottonseed protein concentrate (CPC) on growth, protein utilization and intestinal health of gibel carp (Carassius gibelio, CAS Ⅴ). The FM-based diet was used as the control, and the blended proteins (TMM: CM: CAP: CPC) at ratios of 1 : 1:8 : 2 (BLEND A), 1 : 1:6 : 4 (BLEND B), and 1 : 1:4 : 6 (BLEND C) were used to replace FM at three levels (33%, 67%, 100%), respectively. The results showed that, compared to the control group, growth performance increased significantly when dietary FM was fully replaced by BLEND B (P<0.05), while decreased by BLEND A (P<0.05). The complete substitution of FM with BLEND B significantly upregulated the mRNA expression of intestinal proinflammatory cytokines, anti-inflammatory cytokines, and tight junction-related genes (P<0.05), improving intestinal tissue morphology and health. And it also significantly increased intestinal trypsin activity (P<0.05), upregulated the mRNA expression of amino acid sensory receptor-related and amino acid or peptide transport-related genes (P<0.05), increased protein apparent digestibility coefficient (P<0.05). The 100% substitution of FM with BLEND A significantly upregulated the mRNA expression of intestinal proinflammatory cytokines and downregulated the mRNA expression of anti-inflammatory cytokine il-10 (P <0.05), reduced intestinal villus height (P<0.05), and decreased protein apparent digestibility coefficient (P<0.05). In conclusion, BLEND B could completely substitute dietary FM and was beneficial to the growth and health of gibel carp. Dietary digestible essential amino acids index (DEEAI) was found as an important indicator and should be higher than 79.5% to meet the maximum growth of fish.

Curcumin Suppresses M2 Macrophage-derived Paclitaxel Chemoresistance through Inhibition of PI3K-AKT/STAT3 Signaling.

Breast cancer is the leading cancer in women worldwide. The development of chemoresistance that leads to recurrence and mortality remains a major concern. M2-type tumor-associated macrophages (TAMs), present in the breast tumor microenvironment, secrete various cytokines and growth factors that induce chemoresistance. Curcumin, isolated from Curcuma longa, is known to sensitize cancer cells and increase the efficacy of standard chemotherapeutic agents. However, the effect of curcumin on the chemoresistancegenerating ability of M2 TAMs is not known. The study aimed to determine whether curcumin could modulate M2 macrophages and suppress their ability to induce resistance to paclitaxel in breast cancer cells. THP-1 cells were differentiated to M2 macrophages using PMA and IL-4/IL-13 in the presence or absence of curcumin in vitro. The effect of the conditioned medium of M2 macrophages on inducing resistance towards paclitaxel in MCF-7 or MDA-MB-231 cells was analyzed by cell proliferation assay, cell cycle analysis, wound healing and transwell migration assays. RT-PCR analysis was used to determine the mRNA expression of anti-inflammatory cytokines in M2 macrophages. The effect of curcumin on TGF-β, pAKT, and pSTAT3 in M2 macrophages was analyzed by western blotting. Our data revealed that the M2 macrophages polarized in the presence of curcumin lacked the ability to generate chemoresistance to paclitaxel in breast cancer cell lines. Transcriptomic analysis revealed the expression of TGF-β to be highest amongst M2 macrophage-secreted cytokines. We observed that purified recombinant TGF-β generated chemoresistance in breast cancer cells. We found that curcumin treatment abrogated the expression of TGF-β in M2 macrophages and suppressed their ability to induce chemoresistance in breast cancer cells. STITCH analysis showed strong interaction between curcumin and AKT/STAT3 pathway. Mechanistically, curcumin inhibited PI3K/AKT/STAT3 signaling in M2 macrophages. Western blot analysis revealed that M2 TAM CM, but not curcumin-treated macrophage CM, activated COX2/NF-κB in breast cancer cells. Our results showed that curcumin reduced the chemoresistance-generating ability of M2 TAMs. The study has revealed a non-cancer cell-autonomous mechanism by which curcumin partly overcomes the chemoresistance of paclitaxel in breast cancer.

Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-β) was better than that on the pro-inflammatory cytokines (BpIL-1β and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

Role of sodium octanoate in regulating survival, growth, intestinal development, digestive and absorptive capacities, antioxidant capacity and innate immunity of large yellow croaker (Larimichthys crocea) larvae

The present study was conducted to investigate the effects of dietary sodium octanoate (So) on the survival, growth, intestinal development, digestive and absorptive capacities, antioxidant capacity and innate immunity of Larimichthys crocea larvae. Larvae (initial body weight: 7.44 ± 0.53 mg) were fed with either a control diet or diets with different levels (0.1%, 0.2%, 0.4%, and 0.8%) of So for 30 days. The results showed that larvae fed diets with 0.1%–0.4% So had significantly increased survival compared to the control. Compared to the control, larvae fed the diet with 0.4% So showed dramatically improved growth performance, although there were no significant differences between other diets. Notably, larvae fed diets with 0.2%–0.4% So displayed significantly higher fold height of intestine compared to the control group. Increased levels of selected intestinal cell proliferation and differentiation gene markers (pcna and odc) and increased activity of intestinal brush border enzymes (alkaline phosphatase and leucine aminopeptidase) further affirmed the promotional effect of So on intestinal development. Moreover, for genes related to the intestinal apical junction complex, larvae fed diets with 0.1%–0.8% So showed significantly higher mRNA expression levels of occludin and cateninα1 than the control group. Meanwhile, compared to the control, the expression of claudin1, afadin and cadherin2 significantly increased in larvae fed diets with 0.1%–0.4% So. Also, larvae fed diets with 0.1%–0.8% So showed significantly higher activity of lipase than the control group, whereas the activity of trypsin was significantly higher only in larvae fed the diet with 0.4% So. Furthermore, absorption capacity, as reflected by the activity of brush border enzymes (Na+-K+-Adenosinetriphosphatase and creatine kinase), significantly increased in larvae fed diets with 0.2%–0.4% So compared to the control. Furthermore, larvae fed the diet with 0.4% So exhibited improved antioxidant enzyme activity (catalase and total antioxidant capacity), upregulated antioxidant related gene expression (sod2, sod3, cat, and nrf2), increased antioxidant substance levels (reduced glutathione), and decreased malondialdehyde content compared to the control group. Moreover, larvae fed the diet with 0.4% So showed the highest activity of innate immune-related enzymes (lysozyme, acid phosphatase, inducible nitric oxide synthase and total nitric oxide synthase) and the highest mRNA expression of anti-inflammatory cytokines (il4/13 and arg1). In summary, the diet supplemented with 0.4% So may optimally improve larval survival and growth probably by enhancing intestinal development, digestion and absorption, antioxidant capacity, and innate immunity.

TRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response.

Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown. Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly. Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P<0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1β, P=0.0468; IL-6, P=0.0369) and the macrophages of M1 markers (TNF-α, P=0.0102; CD80, P=0.0056; MCP-1, P=0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P=0.0009; IL-10, P=0.0007; IL-13, P=0.0008) and the mRNA expression of M2 markers (TGF-β1, P=0.0016; ARG1, P=0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P=0.011) but inhibited that of CD206+ macrophages (10.5%, P=0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P=0.0023) and inhibited the expression of Pax7 (P=0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P=0.0002; MyoG, P=0.0037) and myotube formation (21.3%, P=0.0016) but inhibited the positive rate of Edu (27.7%, P=0.0001), cell viability (22.6%, P=0.003) and the number of myoblasts in the G2 phase (26.3%, P=0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-β signalling pathway by targeting Tgfbr1. These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-β signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

Decidual immune response following COVID-19 during pregnancy varies by timing of maternal SARS-CoV-2 infection

While COVID-19 infection during pregnancy is common, fetal transmission is rare, suggesting that intrauterine mechanisms form an effective blockade against SARS-CoV-2. Key among these is the decidual immune environment of the placenta. We hypothesize that decidual leukocytes are altered by maternal SARS-CoV-2 infection in pregnancy and that this decidual immune response is shaped by the timing of infection during gestation. To address this hypothesis, we collected decidua basalis tissues at delivery from women with symptomatic COVID-19 during second (2nd Tri COVID, n = 8) or third trimester (3rd Tri COVID, n = 8) and SARS-CoV-2-negative controls (Control, n = 8). Decidual natural killer (NK) cells, macrophages and T cells were evaluated using quantitative microscopy, and pro- and anti-inflammatory cytokine mRNA expression was evaluated using quantitative reverse transcriptase PCR (qRT-PCR). When compared with the Control group, decidual tissues from 3rd Tri COVID exhibited significantly increased macrophages, NK cells and T cells, whereas 2nd Tri COVID only had significantly increased T cells. In evaluating decidual cytokine expression, we noted that IL-6, IL-8, IL-10 and TNF-α were significantly correlated with macrophage cell abundance. However, in 2nd Tri COVID tissues, there was significant downregulation of IL-6, IL-8, IL-10, and TNF-α. Taken together, these results suggest innate and adaptive immune responses are present at the maternal-fetal interface in maternal SARS-CoV-2 infections late in pregnancy, and that infections earlier in pregnancy show evidence of a resolving immune response. Further studies are warranted to characterize the full scope of intrauterine immune responses in pregnancies affected by maternal COVID-19.

Characterization of large yellow croaker (Larimichthys crocea) osteoprotegerin and its role in the innate immune response against to Vibrio alginolyticus.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily, contributing to inflammation, apoptosis, and differentiation. However, the function of OPG in the host immune system of teleosts remains unclear. Here, we cloned the cDNA of the LcOPG gene from large yellow croaker. LcOPG mRNA was expressed in all analyzed tissues and was upregulated by Vibrio alginolyticus infection in immune tissues and monocytes/macrophages (MO/MФ). Subsequently, the LcOPG protein was expressed and purified using a prokaryotic expression system. Recombinant LcOPG protein (rLcOPG) treatment suppressed V. alginolyticus-induced pro-inflammatory cytokine and enhanced V. alginolyticus-induced anti-inflammatory cytokine mRNA expression. Furthermore, rLcOPG decreased V. alginolyticus-induced MO/MФ apoptosis. Therefore, the results indicate that LcOPG might play a role in the immune response of V. alginolyticus-infected large yellow croaker.

Protective effect and mechanism of nicotinamide adenine dinucleotide against optic neuritis in mice with experimental autoimmune encephalomyelitis

Patients with multiple sclerosis (MS) are commonly accompanied by optic neuritis (ON) that causes retinal ganglion cell (RGC) death and even vision loss. Nicotinamide adenine dinucleotide (NAD+) can protect against cell apoptosis and attenuate MS-triggered symptoms. However, the effect of NAD+ on MS-triggered ON remains unclear. Herein, experimental autoimmune encephalomyelitis (EAE) was established by immunizing female C57BL/6 mice with MOG35-55 peptide. To investigate the effect of NAD+ on ON prevention and treatment, EAE mice received 250 mg/kg NAD+ daily via intraperitoneal injection after immunization and EAE onset, respectively. EX-527 (10 mg/kg, SIRT1 inhibitor) was intraperitoneally injected every two days to explore the role of SIRT1 in NAD+-induced therapeutic effect on EAE. NAD+ intervention attenuated the severity of EAE in mice. NAD+ intervention relieved inflammatory infiltration and CD3+ and CD4+ cell infiltration and decreased the number and activation of microglia and astrocytes in the optic nerve. NAD+ intervention also attenuated demyelination, axonal loss, oligodendrocyte apoptosis and oligodendrocyte progenitor cell recruitment and proliferation in the optic nerve and protected against RGC apoptosis in the retina. NAD+ intervention decreased pro-inflammatory cytokine mRNA and pro-apoptotic protein expression and enhanced anti-inflammatory cytokine mRNA expression and the SIRT1 signaling in the optic nerve and retina and regulated the Th1/Th17/Tregs immune response in the spleen. In addition, EX-527 reversed the therapeutic effect of NAD+ on EAE, suggesting that NAD+ prevented MS-triggered ON by activating the SIRT1 signaling pathway. This study shows the potential of NAD+ to be used as a drug in preventing and treating MS-related ON.

The key role of autophagy in silver nanoparticle-induced BV2 cells inflammation and polarization

As the release of silver nanoparticles (AgNPs) in the environment continues to increase, great concerns have been raised about their potential toxicity to humans. It is urgent to assess the possible toxicity of AgNPs to the immune cells of the central nervous system due to the continuous accumulation of AgNPs in the brain. This study aimed to evaluate the neurotoxicity of AgNPs and the regulatory mechanism of autophagy in AgNPs-induced inflammation by using mouse microglia BV2 cell lines. AgNPs decreased the microglia cell activity in a concentration and time-dependent manner. The exposure of BV2 cells to AgNPs at a non-cytotoxic level of 5 μg/mL resulted in increase of pro-inflammatory cytokines and decrease of mRNA expression of anti-inflammatory cytokines. AgNPs exposure increased M1 markers of iNOS expression and decreased the expression of M2 markers of CD206 in a time-dependent manner. Meanwhile, the expression of inflammatory proteins IL-1β and NF-κB increased significantly. Additionally, AgNPs induced an increase in autophagosome and upregulation of LC3II, Beclin1, and p62 expression levels. Pretreatment by an autophagy inhibitor, 3-Methyladenine, caused more AgNPs-treated microglia to polarized into pro-inflammatory phenotypes. Inhibition of autophagy also increased the expression of inflammation-associated mRNA and proteins in BV2 cells. These results indicated that AgNPs could induce pro-inflammatory phenotypic polarization of microglia and the autophagy could play a key regulatory role in the pro-inflammatory phenotypic polarization of microglia induced by AgNPs.

Comparative assessment of immunomodulatory, proliferative, and antioxidant activities of crocin and crocetin on mesenchymal stem cells.

Saffron (Crocus sativus L) is a well-known spice with active pharmacologic components including crocin, crocetin, safranal, and picrocrocin. Similar to crocin/crocetin, mesenchymal stem cells (MSCs) have been shown to display immunomodulatory and antioxidant properties, which could be beneficial in treatment of various diseases. In the current study, we have evaluated the effects of crocin and crocetin on the functions of MSCs. We used the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay to evaluate MSCs proliferation, and flow cytometry assay to measure the percentage of apoptotic MSCs and Tregs populations. Furthermore, we used the real-time polymerase chain reaction method to quantify messenger RNA (mRNA) expression of inflammatory and anti-inflammatory cytokines. Antioxidant assay was employed to quantify antioxidant parameters including nitric oxide and malondialdehyde levels besides superoxide dismutase activity. Our findings indicated that both crocin and crocetin at low concentrations (2.5 and 5 µM) exhibited significant effects on increasing MSCs viability and on protecting them against apoptosis-induced death. Furthermore, crocin and crocetin at low concentrations (2.5 and 5 µM) displayed a better antioxidant function. Moreover, increased Treg population was observed at lower doses. In addition, crocin/crocetin at low concentrations caused an elevation in mRNA expression of anti-inflammatory cytokines (transforming growth factor-β, interleukin-10 [IL-10], and IL-4), while at higher doses (25 and 50 µM) they led to lowering inflammatory cytokines (IL-1β, IL-6, IL-17, and interferon gamma). Altogether, both crocin and crocetin at lower concentrations exhibited more efficacies on MSCs with a better effect toward crocin. It seems that crocin and crocetin may be considered as complementary treatments for the patients who undergo MSCs transplantation.

Attenuation of mTOR Signaling Is the Major Response Element in the Rescue Pathway of Chronic Kidney Disease in Rats

Background: Modern lifestyle changes and the interlinking of non-communicable diseases result in the development of chronic kidney disease (CKD). While research has focused on attenuating the CKD, the role of mTOR in the progression of CKD is still unclear. Objectives: The current investigation was undertaken to study the role of mTOR-mediated signaling in CKD using Wistar male rats and adenine-induced CKD as an experimental model. Method: The animals were divided into 3 groups, representing control, CKD, and rapamycin-pretreated rats. At the end of the experimental period, blood biochemical indexes on kidney function and expression levels of fibrotic markers, including TGF-β, PAI-1, α-smooth muscle action, fibronectin, CTGF, and collagen-1, were analyzed. In addition, kidney injury markers such as kim-1, cystatin-C, NAG, and NGAL, indicating a progressive fibrotic response, were also studied. Results: The results suggest that mTOR inhibition significantly attenuated the induction of fibrosis, with restored serum creatinine and blood urea nitrogen levels. Intriguingly, the microRNA (miRNA) analysis revealed an increased expression of miR-193–5p, miR-221, miR-212, and miR-183–5p in CKD, while an increased mRNA expression of anti-inflammatory cytokines and reduced level of pS6K with attenuated miRNA was found in rapamycin-treated rats compared to the CKD animals. Conclusion: Activation of mTOR is the major responsive element with activation of miRNAs as an elementary role in the progression of kidney disease. Hence, targeting mTOR would be a possible strategy of treatment for CKD.

Berberis lycium fruit extract attenuates oxi-inflammatory stress and promotes mucosal healing by mitigating NF-κB/c-Jun/MAPKs signalling and augmenting splenic Treg proliferation in a murine model of dextran sulphate sodium-induced ulcerative colitis.

There is a growing interest in developing phytomolecule-based therapies for the management of inflammatory disorders owing to rising cost of treatments and unwarranted effects. The present work attempted to assess the efficacy and mechanisms of Berberis lycium Royle fruit extract (BLFE) in mitigating chemical-induced colitis in mice. Colitis was induced in Balb/C mice using dextran sulphate sodium (DSS) and protective effects of BLFE were examined. Several oxi-inflammatory parameters, histopathological changes, epithelial barrier integrity and activation of NF-κB/c-Jun/MAPKs in colon tissue were determined. Splenic T cell subpopulations were also gauged to evaluate the systemic effects of BLFE in the modulation of immune responses. BLFE treatment effectively improved animal survival rate, DAI score, colon length and structural damage in DSS-exposed mice. Expression of oxi-inflammatory markers such as MPO, IgE, iNOS, ICAM-1, MCP-1 and RANTES as well as Th1/Th2/Th17 cytokines were decreased in BLFE treated animals. On the other hand, an increased mRNA expression of anti-inflammatory cytokines (IL-4/IL-10), tight junction proteins and IgA levels were also observed during BLFE treatment. BLFE appeared to modulate intestinal epithelial cell proliferation (PCNA) and apoptosis (Bcl2/Bax), thereby suggesting its role in the maintenance of intestinal integrity. Analysis of inflammatory signalling pathways indicated robust activation and expression of NF-κB/c-Jun/MAPKs (JNK and p38) in DSS treated animal which was strongly abrogated by BLFE treatment. BLFE supplementation also enhanced the proliferation of CD3+CD4+CD25+ Treg cells indicating suppression of inflammatory activation. These observations provide compelling evidence that BLFE could be considered as a viable natural strategy in the prevention and management of ulcerative colitis.

C-reactive protein/serum amyloid P promotes pro-inflammatory function and induces M1-type polarization of monocytes/macrophages in mudskipper, Boleophthalmus pectinirostris

C-reactive protein (CRP) and serum amyloid P (SAP) play essential roles in the phagocytic cell-mediated innate immune response of mammals. In-depth studies into CRP and SAP have been completed in mammals; however, such studies, particularly those relating to the functions of CRP and SAP, are rare in fish species. In this study, a homolog of CRP/SAP (BpCRP/SAP) was identified in mudskipper (Boleophthalmus pectinirostris), which had the typical characteristics of a fish short pentraxin protein. Phylogenetic tree analysis revealed that BpCRP/SAP was most closely related to mudskipper CRP/SAP-l3. BpCRP/SAP transcripts were detected in all tested tissues, with the highest level observed in the liver; transcripts in the immune tissues and protein expression in the serum were induced in response to Edwardsiella tarda infection. The active recombinant BpCRP/SAP (rBpCRP/SAP) was able to augment the mRNA expression of pro-inflammatory cytokines and attenuate the mRNA expression of anti-inflammatory cytokines in monocytes/macrophages (MO/MΦ). In addition, phagocytosis and bacterial killing of E. tarda by mudskipper MO/MΦ were boosted by rBpCRP/SAP stimulation. rBpCRP/SAP also promoted M1-type MO/MΦ polarization, but inhibited M2-type polarization. In conclusion, the present research describes the pro-inflammatory function of BpCRP/SAP in mudskipper against E. tarda infection.

Changes in Ileal Microbial Composition and Microbial Metabolism by an Early-Life Galacto-Oligosaccharides Intervention in a Neonatal Porcine Model.

Galacto-oligosaccharides (GOS), functional oligosaccharides with natural characteristics, are important active substances in milk that play an important role in the development of intestinal microbiota and the immune system of newborns. The intestinal maturation of piglets resembles that of human newborns and infants. Therefore, we used the newborn piglet model to study the effects of early-life GOS intervention. Six litters of neonatal piglets (10 piglets per litter) with the same average birth weight were divided into control (CON) and GOS (GOS) groups in each litter. Piglets in the GOS group were given 10 mL of GOS solution daily during the first week after birth, while piglets in the CON group were given the same dose of physiological saline orally. One pig per group from each litter was euthanized on day 8 and day 21. Results revealed that ileal microbiota composition was significantly enriched in Lactobacillus and unclassified Lactobacillaceae, and reduced in Clostridium sensu stricto on day 8 and day 21 after GOS intervention. Additionally, Escherichia significantly decreased on day 21 following the early-life GOS intervention. Moreover, the content of microbial metabolites, endocrine peptides, and the mRNA expression of anti-inflammatory cytokines and antimicrobial peptides increased in the GOS group. These findings provide guidelines for early prebiotic supplementation for lactating newborns.

Repurposing simvastatin as a therapy for preterm labor: evidence from preclinical models.

Preterm birth (PTB), the leading cause of neonatal morbidity and mortality, urgently requires novel therapeutic agents. Spontaneous PTB, resulting from preterm labor, is commonly caused by intrauterine infection/inflammation. Statins are well-established, cholesterol-lowering drugs that can reduce inflammation and inhibit vascular smooth muscle contraction. We show that simvastatin reduced the incidence of PTB in a validated intrauterine LPS-induced PTB mouse model, decreased uterine proinflammatory mRNA concentrations (IL-6, Cxcl1, and Ccl2), and reduced serum IL-6 concentration. In human myometrial cells, simvastatin reduced proinflammatory mediator mRNA and protein expression (IL-6 and IL-8) and increased anti-inflammatory cytokine mRNA expression (IL-10 and IL-13). Critically, simvastatin inhibited myometrial cell contraction, basally and during inflammation, and reduced phosphorylated myosin light chain concentration. Supplementation with mevalonate and geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, abolished these anticontractile effects, indicating that the Rho/Rho-associated protein kinase pathway is critically involved. Thus, simvastatin reduces PTB incidence in mice, inhibits myometrial contractions, and exhibits key anti-inflammatory effects, providing a rationale for investigation into the repurposing of statins to treat preterm labor in women.—Boyle, A. K., Rinaldi, S. F., Rossi, A. G., Saunders, P. T. K., Norman, J. E. Repurposing simvastatin as a therapy for preterm labor: evidence from preclinical models.

The Effects of Mouse Recombinant Resistin on mRNA Expression of Proinflammatory and Anti-Inflammatory Cytokines and Heat Shock Protein-70 in Experimental Stroke Model

BackgroundOur recent research showed that resistin has a neuroprotective effect against stroke-induced injury through suppressing apoptosis and oxidative stress. However, the molecular mechanism of neuroprotection of resistin is unclear. This work was designed to examine the effect of mouse recombinant resistin on mRNA expression of Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-10 (IL-10), Transforming growth factor-β1 (TGF- β1), and Heat shock protein-70 (HSP-70) in mouse model of stroke. Materials and MethodsTransient focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) in mice. TNF-α, IL-1β, IL-10, TGF-β1, and HSP-70 mRNA were detected at sham (0 hour), 3 hours, 6 hours, 12 hours, and 24 hours after MCAO using real-time QRT-PCR method. Moreover, animals were treated with resistin at the dose of 400ng/mouse at the commencement of MCAO, and mRNA expression of the cytokines and HSP-70 was measured 24 hours after MCAO. ResultsTumor necrosis factor-α and IL-1β mRNA expression markedly increased at 12-hour time point and then returned to the basal level at 24 hours after MCAO; but HSP-70 mRNA expression increased at 24-hour time point. Furthermore, resistin (400 ng/mouse) significantly increased TGF-β1 and IL-10 and decreased HSP-70 gene expression at 24 hours after MCAO. ConclusionsOur findings revealed that a molecular mechanism of attenuating ischemic damage by resistin administration probably is increased mRNA expression of anti-inflammatory cytokines. However, applying resistin in the clinical settings for the treatment of stroke deserves further researches in the future.

Identification of two IL-4/13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes/macrophages

Mammalian interleukin-4 (IL-4) and −13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1β and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-β and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs.

Immunosuppressive effect of mesenchymal stem cell-derived exosomes on a concanavalin A-induced liver injury model.

BackgroundThis study aimed to evaluate the effect of mesenchymal stem cell (MSC)-derived exosomes on an immune-induced liver injury model. MSCs show a unique function to modulate immune reaction although the molecular mechanisms are still under investigation. Exosomes are a nanoparticle containing microRNA and many ligands and are recognized as important factors secreted from MSC to express their function. This research is undertaken to evaluate the effect of MSC-derived exosome on concanavalin-A (con-A)-induced liver injury.MethodsExosomes were collected from the supernatant of MSC from the bone marrow of C57B6 mice with ultracentrifugation. The collected exosomes or MSCs were injected intravenously into liver injury mice that had been prepared by the intravenous con-A injection. Liver and serum samples were collected 24 h later to evaluate the macro- and microscopic images, the alanine aminotransferase (ALT), and cytokine messenger RNA (mRNA) expression levels. Phenotypical change of non-parenchymal liver cells was also evaluated by flow cytometry. Liver localization of PKH26 after the injection of PKH26-labeled exosomes or MSCs was observed by microscope. Each result was statistically analyzed with Student’s t test.ResultsPKH was observed in the liver after PKH-labeled exosomes were injected into mouse, whereas it was only observed in the lung in a mouse group receiving PKH-leveled MSC. There were decreases in ALT, liver necrotic areas, and the extent of apoptosis indicated by the single-stranded DNA index of groups that received multiple injections of MSC-derived exosomes, but an increase in the Ki-67 index. The mRNA expression of anti-inflammatory cytokines was enhanced. The number of Treg was increased among NPCs in a group receiving exosomes multiple times.ConclusionsSuppression of con-A-induced liver injury by injection of exosomes was observed as same extent as MSC. Considering the advantage of exosomes as its non-living nature and dosing adjustability over MSC, exosome will be one alternative of MSC transplantation.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

BackgroundTolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine.MethodsThe microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests.ResultsWe found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice.ConclusionsMetformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0754-9) contains supplementary material, which is available to authorized users.

MTOR Signaling Regulates Protective Activity of Transferred CD4+Foxp3+ T Cells in Repair of Acute Kidney Injury.

CD4+Foxp3+ regulatory T cells (Tregs) are required for normal immune homeostasis. Recent studies suggested that Treg transfer facilitates recovery from acute kidney injury (AKI), but the molecular events that maintain Treg function after adoptive transfer remain unclear. This study aimed to investigate the regulation of mammalian target of rapamycin (mTOR) signaling in the Treg-mediated therapeutic effect on ischemic AKI. We noted significant Treg expansion in C57BL/6 mouse kidney, with enhanced immunosuppressive capacity after renal ischemia/reperfusion. mTOR inhibition significantly increased the frequency of Tregs in cultured CD4+ T cells, with enhanced production of anti-inflammatory cytokines, which, conversely, was reduced by mTOR activation. Rapamycin, an inhibitor of mTOR, was transiently administered to C57BL/6 mice before ischemia/reperfusion surgery. No beneficial effect of rapamycin treatment was seen in the early recovery of AKI as a result of its inhibitory effect on tubular regeneration. However, rapamycin markedly enhanced the expansion of kidney Tregs, with increased mRNA expression of anti-inflammatory cytokines. Adoptive transfer of rapamycin-treated Tregs markedly suppressed conventional T cells, responder myeloid cells, and reactive myofibroblasts; however, it promoted host Tregs and alternative macrophages, leading to better renal function and less kidney fibrosis. Taken together, Treg transfer with mTOR inhibition markedly improves outcomes of ischemic AKI. These findings reveal an important role for mTOR signaling in maintaining Treg activity after adoptive transfer and highlight the therapeutic potential of targeting Tregs in acute and chronic kidney disease.