TLR4 mRNA Expression Research Articles

L-Isoleucine Administration Alleviates DSS-Induced Colitis by Regulating TLR4/MyD88/NF-κB Pathway in Rats.

Inflammatory bowel disease (namely, colitis) severely impairs human health. Isoleucine is reported to regulate immune function (such as the production of immunoreactive substances). The aim of this study was to investigate whether l-isoleucine administration might alleviate dextran sulfate sodium (DSS)-induced colitis in rats. In the in vitro trial, IEC-18 cells were treated by 4 mmol/L l-isoleucine for 12 h, which relieved the decrease of cell viability that was induced by TNF-α (10 ng/ml) challenge for 24 h (P <0.05). Then, in the in vivo experiment, a total of 44 Wistar rats were allotted into 2 groups that were fed l-isoleucine-supplemented diet and control diet for 35 d. From 15 to 35 d, half of the rats in the 2 groups drank the 4% DSS-adding water. Average daily gain, average daily feed intake and feed conversion of rats were impaired by DSS challenge (P <0.05). Drinking the DSS-supplementing water also increased disease activity index (DAI) and serum urea nitrogen level (P <0.05), shortened colonic length (P <0.05), impaired colonic enterocyte apoptosis, cell cycle, and the ZO-1 mRNA expression (P <0.05), increased the ratio of CD11c-, CD64-, and CD169-positive cells in colon (P <0.05), and induced extensive ulcer, infiltration of inflammatory cells, and collagenous fiber hyperplasia in colon. However, dietary l-isoleucine supplementation attenuated the negative effect of DSS challenge on growth performance (P <0.05), DAI (P <0.05), colonic length and enterocyte apoptosis (P <0.05), and dysfunction of colonic histology, and downregulated the ratio of CD11c-, CD64-, and CD169-positive cells, pro-inflammation cytokines and the mRNA expression of TLR4, MyD88, and NF-κB in the colon of rats (P <0.05). These results suggest that supplementing l-isoleucine in diet improved the DSS-induced growth stunting and colonic damage in rats, which could be associated with the downregulation of inflammation via regulating TLR4/MyD88/NF-κB pathway in colon.

Alleviative effect of melatonin on the decrease of uterine receptivity caused by blood ammonia through ROS/NF-κB pathway in dairy cow

High concentration of blood ammonia can affect the uterus receptivity and decrease fecundity in dairy cow. Melatonin can reduce reactive oxygen species (ROS) level and has antioxidant and anti-inflammatory effects. However, it is not clear whether melatonin can alleviate ammonia-induced apoptosis of endometrial epithelial cell (EEC) and reduced uterus receptivity. The bovine EEC were treated with ammonium chloride and/or melatonin. Cell viability, apoptosis, oxidative stress and mitochondrial membrane potential were measured and the expression of apoptosis-related genes (p53, Cyt-c, Bax, Bcl-2, caspase-8, caspase-9 and caspase-3), uterus receptivity related genes (VEGF, LIF and EGF) and inflammatory factors (TLR-4, IL-6 and NF-κB) were detected. In addition, the expression of VEGF was detected after adding NF-κB inhibitor (40 μM) and IL-6 (1 ng/mL and 50 ng/mL). The results showed that ammonia significantly increased intracellular ROS level, mRNA and protein expression of Bax, p53, Cyt-c, caspase-9, caspase-8, caspase-3, TLR-4, NF-κB and IL-6, promoted cell apoptosis, while decreased mitochondrial membrane potential, the mRNA and protein expression of VEGF and EGF. Interestingly, melatonin significantly mitigated ammonia-induced changes. However, melatonin could not alleviate ammonia-induced changes of IL-6 and VEGF when NF-κB signal pathway was inhibited. The addition of IL-6 significantly reduced mRNA and protein expression of VEGF. In conclusion, ammonia induced EEC apoptosis through ROS production and activation of mitochondrial apoptosis pathway, and induced inflammatory response through TLR4/NF-κB/IL-6 pathway. Melatonin alleviated EEC apoptosis by inhibiting ROS pathway, and reduced IL-6 expression by inhibiting TLR-4/NF-κB signal pathway, which eventually improved VEGF expression and uterus receptivity in dairy cows.

Toll-like receptor inflammatory cascade and the development of diabetic kidney disease in children and adolescents with type 1 diabetes.

This study aimed to evaluate the association of toll-like receptor (TLR) inflammatory cascade with the development of diabetic kidney disease (DKD) in children and adolescents with type 1 diabetes (T1D). A total of 49 T1D patients and 49 normoglycaemic (NG) subjects aged 5-20 years old were recruited. TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 mRNA expressions were measured in peripheral blood mononuclear cells by reverse transcription-quantitative polymerase chain reaction. Fasting glucose, glycated haemoglobin, serum urea, serum creatinine and urinary albumin-to-creatinine ratio (ACR) were determined. The mRNA expressions of TLR2, TLR4, MYD88 and NFKB were significantly increased in the T1D group compared with the NG group. The mRNA expression levels of MCP1/CCL2 and IL18 were higher in 21 T1D patients (42.9%) (average of MCP1/CCL2: 6.6-fold and IL18: 5.8-fold) than in NG patients. Furthermore, ACR was increased in the T1D group compared with the NG group. The increased mRNA expression of TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 favours the development of an inflammatory process that may lead to a decline in renal function and consequently DKD in children and adolescents with T1D. This suggests that these genes are early mediators of onset DKD since the beginning of the lives of the paediatric T1D patients.

Yeast cell wall upregulated cell-mediated immune responses to Newcastle disease virus vaccine

A recent study has suggested that yeast cell wall product (YP) enhanced serum hemagglutination inhibition (HI) titers and intestinal sIgA responses in chickens immunized with Newcastle disease virus (NDV) vaccine. In the present study, the cell-mediated immune responses elicited by NDV and YP were investigated in commercial broilers. Broilers were fed 0 or 0.1% YP and immunized with a live NDV vaccine via an intraocular-and-intranasal route at 14 and 28 days old. After that, blood samples were collected for determination of HI titer, cytokine content, and blood analysis. Eight chickens were randomly selected from each group and sacrificed. Lymphocytes were harvested from the spleens for lymphocyte proliferation and flow cytometry analysis. Total RNA was extracted from spleen and jejunum for RT-qPCR analysis. The results showed that YP significantly increased serum concentration of IL-4, IL-6, IFN-γ, TNF-β, as well as promoted lymphocytes proliferation in broilers immunized with NDV vaccine. The enhanced cell-mediated immunity is correlated with the upregulated mRNA expression of TGF-β, IL-6, TLR5, GATA-3, and T-bet in the spleen and upregulated mRNA expression of CCR-9, J-chain, pIgR, and TLR3 in the jejunum of chickens. It is noteworthy that no significant side effect was observed after the administration of YP. Therefore, YP could be safely used as potential immunopotentiator assisting NDV vaccine for chickens.

Bacillus subtilis BN strain promotes Th1 response via Toll-like receptor 2 in polarized mouse M1 macrophage.

Bacillus subtilis BN strain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The present study investigated the Th1 responses of the BN strain on a mouse macrophage cell line, J774.1. In cell cultures, the BN strain (spore cell cultured in Schaeffer's sporulation media) significantly increased the production of interleukin (IL-)12 protein. The BN strain induced the mRNA expression of M1 polarization genes, such as inducible nitric oxide synthase and IL-12p40 mRNA, and suppressed the mRNA expression of intracellular marker genes of M2 polarization, such as arginase 1 mRNA. The BN strain downregulated the mRNA expression of Toll-like receptor 4 (TLR4), while it upregulated the mRNA expression of TLR2, MyD88, and nuclear factor kappa B (NF-κB). The production of IL-12 protein induced by the BN strain was decreased by inhibitors of MyD88, NF-κB, and IκB kinase. Moreover, the production of IL-12 was strongly suppressed by neutralizing antibody against TLR2. These results suggest that the BN strain promotes Th1 response via TLR2 signal in mouse M1 macrophage. PRACTICAL APPLICATIONS: Bacillus subtilis is known to have beneficial effects for the host. B. subtilis BN stain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The effects of the BN strain on the Th1 response in macrophage cell cultures were investigated in this work. We found that the spore cells of BN strain promoted the production of Th1-type cytokine, and induced macrophage M1 polarization via Toll-like receptor 2. This study can serve as a significant reference for the development of functional food and feed with immunostimulatory effects. Over time, new food and feed products containing the BN strain may emerge, such as Juice, powder, and tablet.

Intranasal curcumin and dexamethasone combination ameliorates inflammasome (NLRP3) activation in lipopolysachharide exposed asthma exacerbations

The inflammasome NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) is closely associated with exacerbation of asthma as endotoxin (lipopolysaccharide, LPS) is one of its activators present in the environment. Present study is undertaken to investigate anti-inflammatory effects of a well known phytochemical, curcumin, which might regulate LPS exposed asthma exacerbations by modulating NLRP3 activation if given through intranasal route. Balb/c mice were sensitized with intraperitoneal injection of OVA (Ovalbumin; 100 μg of OVA with alum) from day 1 to 8 and exposed to LPS with 1% OVA aerosol from day 9 to 15. LPS (0.1 μg) was given an hour before sensitization and OVA-aerosol challenge. Significant decrease in inflammatory cell recruitment and restoration of structural changes in lungs, alterations in mRNA and protein expressions of TLR-4, NF-κB, NLRP3, Caspase-1, IL-1β, MMP-9, IL-5 and IL-17 in intranasal curcumin alone and corticosteroid combined pretreatment group.

Syringin protects against cerebral ischemia and reperfusion injury via suppression of inflammatory mediators and toll-like receptor/MyD88 signaling pathway in rats

Background: The cerebral ischemic stroke befalls in response to the abrupt occlusion of blood vessels that leads to deprived oxygen and glucose in brain and causes brain injuries. Stroke is associated with higher morbidity and mortality rate globally. Syringin is a bioactive compound from Eleutherococcus senticosus and possesses plentiful biological actions. Objectives: In this research work, we envisioned to observe the neuroprotective actions of syringin against ischemic-reperfusion (I/R)-provoked ischemic stroke in rats. Materials and Methods: The focal cerebral I/R injuries were roused to the male Wistar rats through the middle coronary artery occlusion (MCAO) technique. The syringin (10, 25, and 50 mg/kg) was administered to the rats through intragastric route for 7 successive days prior to MCAO and another 3 days after MCAO. Sham rats were administered with usual diet. The neurological score and parameters were measured by standard methods. The status of inflammatory cytokines and mediators in both serum and brain tissues was considered by commercial assay kits. The mRNA expression of toll-like receptor (TLR) 4, MyD88, Fas, and FasL was inspected by reverse transcription–polymerase chain reaction technique. Results: The syringin administration to I/R rats established the lessened neurological score and parameters. Syringin supplementation was meritoriously suppressed the levels of the inflammatory cytokine, i.e., interleukin (IL)-1 β, IL-6, and tumor necrosis factor-α and enhanced the IL-10 status in I/R rats. Syringin abridged the inflammatory mediators like cyclooxygenase-2, prostaglandin-2, and nuclear factor kappa B levels in the serum and brain tissues. The mRNA expression of TLR4, MyD88, Fas, and FasL was noticeably downregulated by the syringin. Conclusion: Taken together, our results showed the curative role of syringin against ischemic stroke in rats. Syringin can be an auspicious anti-stroke agent in future to treat ischemic stroke.

Expression of Toll-Like Receptors 2, 4 and 5 in Relation to Gut Microbiota in Colon Neoplasm Patients with and without Inflammatory Bowel Disease.

Background:Toll-Like Receptors (TLRs) are the critical mediators of inflammatory routs in the gut, which play an essential role in regulating the immune responses towards various ligands derived from pathogenic bacteria. Also, TLR signaling has been implicated in the development of Inflammatory Bowel Disease (IBD), Adenomatous Polyp (AP), and Colorectal Cancer (CRC). Here, we aimed to examine the expression of some TLRs concerning certain fecal bacteria in AP and CRC patients with and without IBD.Methods:This case-control study collected fecal and colonic tissue samples from 93 patients versus Normal Controls (NC) via colonoscopy. Fecal samples were used for DNA extraction, and the abundance of selected fecal bacteria was determined by absolute real-time PCR. Also, the gene expression of TLR2, 4, and 5 was analyzed using RT-PCR on the colonic tissues of participants.Results:Compared to NC individuals, in AP and CRC patients, the mRNA expressions of TLR4 and TLR2 were significantly increased while TLR5 was decreased. A meaningful association between TLRs mRNA expression levels and the abundance of some selected fecal bacteria was detected. Also, there was a significant relationship between participant’s food regimes, smoking habit and intestinal TLRs expression.Conclusion:Our study proposed the important role of TLRs during adenomatous and CRC formation. Alterations in TLRs expression associated with certain gut bacteria may contribute to disease development.

Mechanism of dexmedetomidine preconditioning on spinal cord analgesia in rats with functional chronic visceral pain

ABSTRACTPurpose:To analyze the effect and mechanism of dexmedetomidine (DEX) analgesia pretreatment on functional chronic visceral pain in rats.Methods:Rats were divided into six groups: W1, W2, W3, W4, W5, and W6. The behavioral changes and electrophysiological indexes of rats in each group before and after DEX treatment were detected.Results:The levels of abdominal withdrawal reflex (AWR) in W5 and W6 groups were significantly lower than those in group W3, while the levels of thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were significantly higher than those in group W3 (p < 0.05). The electromyographic signals of W1, W5, and W6 groups showed little fluctuation, while those of groups W2, W3, and W4 showed obvious fluctuation. TLR4 mRNA expression, IRF3, P65, and phosphorylation levels in W4, W5, and W6 groups were significantly lower than those in group W2 (p < 0.05).Conclusions:Dexmedetomidine epidural anesthesia pretreatment could significantly inhibit visceral pain response in rats with functional chronic visceral pain, and its mechanism was related to the activation of TLR4 in spinal dorsal horn tissue of rats and the activation inhibition of IRF3 and P65 in the downstream key signals.

Fecal Microbial Enterotypes Differentially Respond to a High-fat Diet Based on Sex in Fischer-344 Rats.

The gut microbiota interacts with the host gut environment, which is influenced by such factors as sex, age, and host diet. These factors induce changes in the microbial composition. The aim of this study was to identify differences in the gut microbiome of Fisher-344 (F344) rats fed a high-fat diet (HFD), depending on their age and sex. Fecal microbiomes from 6-, 31-, and 74-week-old, and 2-year-old both male and female rats (corresponding to 5-, 30-, 60-, and 80-year-old humans) were analyzed using 16S rRNA gene sequencing, phylogenetic investigation of communities by reconstruction of unobserved states, and enterotype (E) assessment. Moreover, the effect of an HFD on colonic epithelial cells was measured using real-time quantitative PCR. Alpha diversity decreased in the HFD group regardless of age and sex. Based on the enterotype clustering of the whole fecal microbiome, clusters from male rats were divided into E1 and E2 enterotypes, while clusters from female rats were divided into E1, E2, and E3 enterotypes. The female E3 group showed a significantly high abundance in the Ruminococcus genus and expression of Tlr2 mRNA, which may reflect compensation to the HFD. Moreover, the female E3 group showed a lower ratio of opportunistic pathogenic strains to commensal strains compared to the female E2 group. Administration of an HFD influenced the rat fecal microbiota in all assessed age groups, which could be further differentiated by sex. In particular, female rats showed a compensatory enterotype response to an HFD compared to male rats.

Alterations in Toll-Like Receptor 7 and -9 mRNA Levels in Lungs after Ovariohysterectomy in a Pyometra Mouse Model

Background: Pyometra (P) leads to sepsis and multiple organ dysfunction syndrome. Toll-like receptors (TLRs) recognize pathogens which can cause P. The aim of this study was to investigate TLR-7 and -9 via the MYD88 pathway and the nuclear factor kappa B (NFκB) response in the uterus of a P mouse model before and after ovariohysterectomy (RP) as well as potential lung injury. Materials and Methods: 200 female C57BL/6J mice were randomly divided into groups (N = 10/subgroup; sham 1, 2, 3, 7; P1, 2, 3, 7; 1RP1, 2, 3, 7; 2RP1, 2, 3, 7; 3RP1, 2, 3, 7) according to the day of euthanasia. Pathogens were administrated in the groups P and RP in order to induce P. Results: Alterations in blood chemistry, histopathology, and RT-qPCT analysis before (P) and after RP were observed. Significant correlations were also found between MYD88, NFκB, and TLR9 in P and RP groups in the lungs and in RP groups in the uterus, suggesting that the immune system responded via the TLR9-MYD88 pathway. Conclusions: This is the first report of immunohistochemical TLR-7 and -9 localization and of TLR-7, -9, MYD88, and NFκB mRNA expression in the uterus causing lung injury in a P mouse model.

Immunosuppressive effect of Columbianadin on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced dendritic cells

Ethnopharmacological relevanceAngelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was unclear. Columbianadin (CBN) was one of the main bioactive compounds of APR, and has many pharmacological effects. But the immunosuppressive effect of CBN on DCs and the potential mechanism needed to be explored. Aim of the studyThe study was aimed to clarify the immunosuppressive effect of CBN on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced DCs. Materials and methodsBone marrow-derived DCs were obtained and cultured from C57BL/6 mice in accordance with protocol. The phenotypic study (CD11c, CD40, CD80, CD86 and MHC Ⅱ) were measured by flow cytometry. FITC-dextran were uptaked by DCs and the change of endocytosis activity were mediated by acquired mannose receptor. Transwell chambers were used to detect the migration ability of DCs. Mixed leukocyte reaction (MLR) assay was used to detect the allostimulatory ability of CBN on TNF-α stimulated DCs. The secretion of cytokines and chemokines was measured by ELISA Kit. TLRs gene and MAPKs/NF-κB protein expression were checked by qRT-PCR and Western blot. ResultsCBN inhibited the maturation of TNF-α-induced DCs while maintaining phagocytosis capabilities. Additionally, CBN inhibited the migration of TNF-α stimulated DCs, which related to reduce the production of chemokines (MCP-1, MIP-1α). Notably, CBN could suppress the proliferation of CD4+T cells by inhibiting DCs maturation, and decrease the proinflammatory cytokines IL-6 production. Furthermore, CBN inhibited mRNA expression of TLR2, TLR7 and TLR9 in TNF-α-activated DCs. Meanwhile, the phosphorylation of p38, JNK1/2 and NF-κB protein were significantly inhibited in CBN treated DCs. ConclusionsThese findings provided novel insights into the pharmacological activity of CBN. They also indicated that inhibition DCs maturation owning to the immunosuppressive effect of CBN. CBN was expected as a potential immunosuppressant and TLRs/MAPKs/NF-κB pathway may be an important mechanism for CBN's immunosuppressive activity.

High mobility group box-1 (HMGB-1) and its receptors in the pathogenesis of malaria-associated acute lung injury/acute respiratory distress syndrome in a mouse model

The DNA-binding protein high mobility group box-1 (HMGB-1) mediates proinflammatory cytokines that contribute to acute lung injury (ALI). Although ALI is a frequent complication of malaria infection, the contribution of HMGB-1 and its receptors to the pathogenesis of malaria-associated ALI/acute respiratory distress syndrome (MA-ALI/ARDS) has not been investigated in a mouse model. Here, the malaria-infected mice were divided into two groups according to lung injury score: the ALI/ARDS and non-ALI/ARDS groups. The expression of HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) in lung tissues was investigated by using immunohistochemical staining and real-time polymerase chain reaction (PCR). Additionally, HMGB-1 and proinflammatory cytokine (TNF-α, IFN-γ, IL-1 and IL-6) levels in plasma and lung tissues were quantified by using enzyme-linked immunosorbent assays. Cellular expression of both HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) was significantly increased in the lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups. The levels of HMGB-1, TNF-α, IFN-γ, IL-1 and IL-6 were significantly increased in both plasma and lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups, which were similar to the results obtained by real-time PCR. Increased mRNA expression of RAGE, TLR-2 and TLR-4 was found in the lung tissues of the ALI/ARDS group. Furthermore, the plasma HMGB-1 level was positively correlated with TLR-4 mRNA expression in the ALI/ARDS group. HMGB-1 levels were significantly increased in plasma and lung tissues of MA-ALI/ARDS mice and were related to the upregulated expression of HMGB-1 and proinflammatory cytokines. In conclusion, this study demonstrates that HMGB-1 is an important mediator of MA-ALI/ARDS pathogenesis and may represent a target for therapeutic malaria interventions with ALI/ARDS.

Polysaccharide fraction isolated from the leaves of Hordeum vulgare L. protects against colonic inflammation of systemic immune responses

• BLE0 ameliorated LPS-induced endotoxemia and colonic inflammation. • BLE0 exerted anti-inflammatory activities by suppressing the activation of NF-κB and AP-1 pathways. • BLE0 inhibited the expression of TLR4, Myd88, IRAK1, and TRIF. Sepsis is a life-threatening condition that can result in death accompanied by multiple organ failure. Here, we investigated the preventive effects of BLE0, a polysaccharide fraction isolated from young barley leaves ( Hordeum vulgare L.), on colonic inflammation in a mouse model of LPS-induced systemic septic shock. Pretreatment with BLE0 increased the survival rate and markedly restored colon length and crypt damage in LPS-challenged mice. BLE0 suppressed the LPS-induced expression of iNOS, COX-2, IL-6, and IL-17. The molecular mechanisms underlying the anti-inflammatory effects of BLE0 included inactivation of NF-κB and AP-1 and inhibition of monocyte/macrophage infiltration. BLE0 inhibited the mRNA expression of TLR4, Myd88, IRAK1, and TRIF induced by LPS. Collectively, BLE0 ameliorated LPS-induced endotoxemia and colonic inflammation, suggesting thatt the regulation of colonic conditions could be associated with improved systemic immune responses.

The Suppressive Effect of Mamiran Cream on Atopic Dermatitis-Like Skin Lesions In Vivo.

Background The Chinese herbal formula Mamiran cream (MMC) has been known for its ameliorative effects on diverse skin diseases, such as eczema. Atopic dermatitis (AD; eczema) is a chronic recurrent skin disease dominated by T-helper type 2-driven inflammation (Th2). Objective In this study, the inhibitory effect of MMC on AD was investigated in vivo. Methods An animal model was established by sensitization with 2,4-dinitrochlorobenzene (DNCB) on the skin of SD rats. Cutaneous administration of MMC was applied, and its mechanism of action was investigated via RT-PCR and IHC assay. Result Our data showed that topical application of MMC reduced the skin severity scores and alleviated the histological changes. Furthermore, immunohistochemical analysis demonstrated that MMC significantly decreased the levels of Th2 cytokine IL-5 and IL-4Ra in the skin lesion. In addition, it was demonstrated that MMC downregulated the mRNA expression of TNF-α, IL-1β, IL-6, IL-10, and TLR4. Moreover, MMC inhibited the activation of NF-κB, JNK1, and STAT6 pathways in skin lesions. Conclusions Our findings suggest that MMC exhibits the inhibitory effect on AD, suggesting that MMC may be a potential therapeutic agent for this atopic disorder.

Dietary Enteromorpha Polysaccharide Enhances Intestinal Immune Response, Integrity, and Caecal Microbial Activity of Broiler Chickens.

Marine algae polysaccharides have been shown to regulate various biological activities, such as immune modulation, antioxidant, antidiabetic, and hypolipidemic. However, litter is known about the interaction of these polysaccharides with the gut microbiota. This study aimed to evaluate the effects of marine algae Enteromorpha (Ulva) prolifera polysaccharide (EP) supplementation on growth performance, immune response, and caecal microbiota of broiler chickens. A total of 200 1-day-old Ross-308 broiler chickens were randomly divided into two treatment groups with ten replications of ten chickens in each replication. The dietary treatments consisted of the control group (fed basal diet), and EP group (received diet supplemented with 400 mg EP/kg diet). Results showed that chickens fed EP exhibited significantly higher (P < 0.05) body weight and average daily gain than the chicken-fed basal diet. In addition, significantly longer villus height, shorter crypt depth, and higher villus height to crypt depth ratio were observed in the jejunal and ileal tissues of chickens fed EP. EP supplementation upregulated the mRNA expression of NF-κB, TLR4, MyD88, IL-2, IFN-α, and IL-1β in the ileal and jejunal tissues (P < 0.05). Besides, we observed significantly higher (P < 0.05) short-chain volatile fatty acids (SCFAs) levels in the caecal contents of the EP group than in the control group. Furthermore, 16S-rRNA analysis revealed that EP supplementation altered gut microbiota and caused an abundance shift at the phylum and genus level in broiler chicken. Interestingly, we observed an association between microbiota and SCFAs production. Overall, this study demonstrated that supplementation of diet with EP promotes growth performance, improves intestinal immune response and integrity, and modulates the caecal microbiota of broiler chickens. This study highlighted the application of marine algae polysaccharides as an antibiotic alternative for chickens. Furthermore, it provides insight to develop marine algae polysaccharide-based functional food and therapeutic agent.

Crocetin attenuating Urinary tract Infection and adherence of uropathogenic E. coli in NRK-52E cells via an inflammatory pathway.

Urinary tract infections (UTI) are commonly treated with broad-spectrum antibiotics, but treatment has limitation due to causes of nephrotoxicity in uroepithelial cells. Recently, the researcher focuses their research on alternative therapy for the treatment of UTI. This study evaluated the anti-infectious effect of crocetin against adherence of pathogenic [2-14 C]-acetate labeled Escherichia coli (MTCC-729) to rat proximal renal tubular cells (NRK-52E cells) and explores the possible mechanism of action. In vitro cytotoxicity and radio acetate labeled tests were performed on NRK-52E cells. The rats were divided into five different groups as follows: normal control (NC), disease control (DC), and various doses of crocetin (1.25, 2.5, and 5mg/kg) treated group rats. White blood cells in blood, urine, and bacterial colony counts were estimated at regular intervals. Pro-inflammatory cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), and interleukin-8 (IL-8), were also estimated. In the current study, we estimated the mRNA expression of toll-like receptor-4 (TLR-4) and toll-like receptor-2 (TLR-2) in the renal and bladder tissues. Crocetin significantly (p<.05) inhibited the adherence of E. Coli in NRK-52E cells. Crocetin suppresses the lipid peroxidation (LPO) 42% in cells treated with H2 O2 cells without crocetin. The white blood cells (WBC) count in blood and urine were augmented and crocetin treatment significantly (p<.05) reduced the WBC in urine and blood. The pro-inflammatory cytokines, such as IL-6, MCP-1, IL-10, and IL-8, significantly (p<.05) increased in the DC group and crocetin significantly (p<.05) reduced the pro-inflammatory cytokines. Dose-dependent treatment of crocetin significantly reduced the mRNA expression of TLR2 and TLR4 in the renal and bladder tissues. Crocetin considerably reduced the bacterial adherence to NRK-52E cells, attenuated the H2 O2 induced toxicity in NRK-52E cells and also improved the renal tubular function, and reduced the inflammatory response via altering the inflammatory and antioxidant markers. As we all know that urinary tract infection is the most common disease worldwide. In this study, we scrutinized the protective effect of crocetin against urinary tract infection. Crocetin treatment considerably reduced the zone of inhibition and improved radioactivity. Crocetin significantly reduced the levels of cytokines and inflammatory mediators. Crocetin can be used as a protective drug in the treatment of urinary tract infections.

The effect of miR-1338 on the immunomodulatory activity of ophiopogon polysaccharide liposome

This study is to investigate the effect of microRNA-1338 (miR-1338) on the activity of Kupffer cells (KCs) and its mechanism regulated by ophiopogon polysaccharide liposome (OPL). KCs was treated with different OPL after transfected with miR-1338 mimic and miR-1338 inhibitor. The secretion of NO and iNOS, the expression of catalase (CAT) and peroxidase (POD), the phagocytic activity, the expression of CD14 and MHC II, the apoptosis and the secretion of ROS were measured. In addition, the expressions of key signal factors TLR4, IKKβ, MyD88 and NF-κB in NF-κB signaling pathway were measured by real-time PCR and Western blot (WB). The results showed that OPL could promote the secretion of iNOS, the expression of POD, the phagocytosis, the mRNA expression of TLR4, MyD88, IKKβ and NF-κB, the protein expression of TLR4 and NF-κB, and inhibit the cell apoptosis and ROS secretion after transfected with miR-1338 mimic. After transfected with miR-1338 inhibitor, OPL could promote the secretion of NO and iNOS, the expression of POD, cell migration, phagocytosis, and inhibit cell apoptosis. Meanwhile, the mRNA expression of TLR4, MyD88, IKKβ and NF-κB and the protein expression of TLR4, MyD88 and NF-κB were promoted. These results suggested that OPL could activate TLR4-NF-κB signaling pathway and thereby improve the activity of KCs by regulating miR-1338.

In vivo and in vitro studies of Danzhi Jiangtang capsules against diabetic cardiomyopathy via TLR4/MyD88/NF-κB signaling pathway

ObjectivesDanzhi Jiangtang capsule (DJC) is widely used for preventing and treating diabetic cardiomyopathy (DCM). However, the underlying mechanisms of the anti-inflammatory and antiapoptotic activities are unclear. MethodsIn the in vivo diabetic cardiomyopathy rat model, cardiac function was measured through echocardiography, histological changes in the myocardium were visualized using HE staining, and cardiomyocyte apoptosis was detected using TUNEL. The serum levels of anti-inflammatory cytokines were detected using ELISA. Finally, TLR4, MyD88, and NF-κB mRNA expressions were analyzed using RT-qPCR. In the in vitro experiments, the apoptosis rate of the H9c2 cells was detected using FCM; moreover, TLR4, MyD88 and NF-κB mRNA expressions were measured using RT-qPCR and related protein levels were investigated using Western blotting. ResultsIn vivo, DJC effectively improved cardiac function, alleviated the pathological changes, and reduced the apoptosis rate. Moreover, DJC reduced TNF-α, IL-1β, and IL-6 activities, with significant inhibition of the TLR4, MyD88 and NF-κB p65 mRNA expression. Moreover, in vitro, DJC effectively inhibited high-glucose-induced H9c2 apoptosis-an effect similar to that for TAK242. Finally, both the DJC and TAK242 considerably reduced TLR4, MyD88, NF-κB, Bax, and caspase-3 protein expression but increased that of BCL-2. ConclusionsDJC prevented the overactivation of the TLR4/MyD88/NF-κB signaling pathway and regulate cardiomyocyte apoptosis against DCM.

Geraniol Ameliorates Sepsis in Neonatal Rats by Regulating Calprotectin and Inflammatory Response

Sepsis is characterized by systemic infection that causes multiple organ dysfunction and is associated with a high mortality rate in the paediatric intensive care unit. In the present study, we investigated the effect of geraniol on sepsis in neonatal rats. Sepsis was induced in neonatal rats by intraperitoneal (i.p.) inoculation with almost 6,000 colony forming units of Escherichia coli. The rats were treated with geraniol (25 or 50 mg/kg i.p.) 1 h after the administration of E. coli. Survival rate over 30 h, bacterial load, blood parameters, and inflammatory cytokines were measured. Histopathological analysis and qRT-PCR were performed on rat liver tissues. Geraniol improved the survival rate and sepsis severity in neonatal sepsis rats. Inflammatory cytokine levels, liver function test results, and blood parameters were improved in the geraniol-treated compared to sepsis group. Geraniol attenuated mRNA expression of calprotectin (S100A8/S100A9) and TLR-4 in the liver tissue of neonatal sepsis rats. Therefore, geraniol reduced bacterial load in body fluids and inflammatory mediators in neonatal sepsis rats by inhibiting calprotectin and TLR-4 pathways.