Safflower polysaccharide ameliorates acute ulcerative colitis by regulating STAT3/NF-κB signaling pathways and repairing intestinal barrier function.

This study is to investigate the effect of SPS on the UC model. An animal model of UC induced by DSS was developed using C57BL/6 mice. The body weight was recorded every day, and the symptoms related to UC were detected. H&E staining, AB-PAS staining and PSR staining were used to evaluate the histopathological changes of the colon. Inflammation and mucosal barrier indicators were detected by qRT-PCR, and the 16 S rRNA sequence was used to detect the intestinal flora. SPS can significantly prevent and treat DSS-induced ulcerative colitis in animals. SPS significantly improved clinical symptoms, alleviated pathological damage, inhibited the infiltration of intestinal inflammatory cells. SPS treatment can protect goblet cells, enhance the expression of tight junction proteins and mucins, inhibit the expression of antimicrobial peptides, thereby improving intestinal barrier integrity. The prevention and treatment mechanism of SPS may be related to the inhibition of STAT3/NF-κB signaling pathway to regulate intestinal barrier function. In particular, SPS also significantly adjusted the structure of intestinal flora, significantly increasing the abundance of Akkermansia and Limosilactobacillus and inhibiting the abundance of Bacteroides. Overall, SPS has a significant therapeutic effect on ulcerative colitis mice, and is expected to play its value effectively in clinical treatment.

Isoliquiritigenin alleviates the development of alcoholic liver fibrosis by inhibiting ANXA2.

The study aimed to investigate the effect of isoliquiritigenin (ISL) on model of alcoholic liver fibrosis (ALF). C57BL/6 mice were used to establish animal model of ALF, HSC-T6 cells were used to establish alcohol-activated cell model, and tandem mass tag (TMT) assays were used to analyze the proteome. The results showed that ISL obviously alleviated hepatic fibrosis in model mice. ISL visually improved the area of liver pathological stasis and deposition of fibrillar collagen (Sirius Red staining, Masson staining), inhibited the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) in liver tissues. ISL down-regulated the mRNA expression levels of IL-6 and transforming growth factor-ß1(TGF-ß1) in activated hepatic stellate cells (HSCs). And ISL significantly reduced annexin A2 (ANXA2) in vitro detected by TMT proteomics technology. Interestingly, it was found for the first time that ISL could inhibit ANXA2 expression both in vivo and in vitro, block the sphingosine kinases (SPHKs)/sphingosine-1-phosphate (S1P)/interleukin 17 (IL-17) signaling pathway and regulate the expression of α-smooth muscle actin (α-SMA) by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at the downstream signal to finally reverse HSCs activation and hepatic fibrosis. Thus, we demonstrated that ISL is a drug monomer with notable anti-hepatic fibrosis activity.

Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection.

It is not clear how the complex interactions between diet and intestinal immune cells protect the gut from infection. Neutral ceramidase (NcDase) plays a critical role in digesting dietary sphingolipids. We find that NcDase is an essential factor that controls intestinal immune cell dynamics. Mice lacking NcDase have reduced cluster of differentiation (CD) 8αß+ T cells and interferon (IFN)-γ+ T cells and increased macrophages in the intestine and fail to clear bacteria after Citrobacter rodentium infection. Mechanistically, cellular NcDase or extracellular vesicle (EV)-related NcDase generates sphingosine, which promotes macrophage-driven Th1 immunity. Loss of NcDase influences sphingosine-controlled glycolytic metabolism in macrophages, which regulates the bactericidal activity of macrophages. Importantly, administration of dietary sphingomyelin and genetic deletion or pharmacological inhibition of SphK1 can protect against C. rodentium infection. Our findings demonstrate that sphingosine profoundly alters macrophage glycolytic metabolism, leading to intestinal macrophage activation and T cell polarization, which prevent pathogen colonization of the gut.

Inhibition of Sphingosine-1-Phosphate-Induced Th17 Cells Ameliorates Alcohol-Associated Steatohepatitis in Mice.

BACKGROUND AND AIMS: Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to the pathogenesis of alcohol-associated liver disease (ALD). APPROACH AND RESULTS: We used wild-type mice, retinoid-related orphan receptor gamma t (RORγt)-deficient mice, sphingosine kinase-deficient mice, and local gut anti-inflammatory, 5-aminosalicyclic acid-treated mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. Alcohol intake induces a pro-inflammatory shift in immune cell populations in the gut, including an increase in Th17 cells. Using RORγt-deficient mice, we found that Th17 cells are required for alcohol-associated gut inflammation and the development of ALD. Treatment with 5-aminosalicyclic acid decreases alcohol-induced liver injury and reverses gut inflammation by the suppression of CD4+ /RORγt+ /interleukin-17A+ cells. Increased Th17 cells were due to up-regulation of sphingosine kinase 1 activity and RORγt activation. We found that S1P/S1PR1 signaling is required for the development of Th17 cell-mediated ALD. Importantly, in vivo intervention blocking of S1P/S1PR1 signaling markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. CONCLUSIONS: Gut inflammation is a functional alteration of immune cells in ALD. Reducing gut Th17 cells leads to reduced liver damage. S1P signaling was crucial in the pathogenesis of ALD in a Th17 cell-dependent manner. Furthermore, our findings suggest that compounds that reduce gut inflammation locally may represent a unique targeted approach in the treatment of ALD.

Neutral Ceramidase Mediates Nonalcoholic Steatohepatitis by Regulating Monounsaturated Fatty Acids and Gut IgA+ B Cells.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is associated with obesity and an increased risk for liver cirrhosis and cancer. Neutral ceramidase (NcDase), which is highly expressed in the intestinal brush border of the small intestine, plays a critical role in digesting dietary sphingolipids (ceramide) to regulate the balance of sphingosine and free fatty acids. It remains unresolved whether obesity-associated alteration of NcDase contributes to the manifestation of NASH. Here, we revealed that NcDase deficiency in murine models of NASH prevents hepatic inflammation and fibrosis but not steatosis. APPROACH AND RESULTS: NcDase-/- mice display reduced stearoyl-CoA desaturase (SCD) 1 expression with a compositional decrease of monounsaturated fatty acids (MUFAs) under the different dietary conditions. We further found that NcDase is a functional regulator of intestinal B cells and influences the abundance and quality of the secretory IgA response toward commensal bacteria. Analysis of composition of the gut microbiota found that Clostridiales colonization was increased in NcDase-/- mice. The colonization of germ-free mice with gut microbiota from NcDase-/- mice resulted in a greater decrease in the expression of SCD1 and the level of MUFAs in the liver relative to gut microbiota from wild-type littermates, which are associated with the alternation of IgA-bound bacteria, including increase of Ruminococcaceae and reduction of Desulfovibrio. Mechanistically, NcDase is a crucial link that controls the expression of SCD1 and MUFA-mediated activation of the Wnt/ß-catenin. Very importantly, our experiments further demonstrated that Wnt3a stimulation can enhance the activity of NcDase in hepatocytes. CONCLUSIONS: Thus, the NcDase-SCD1-Wnt feedback loop promotes the diet-induced steatohepatitis and fibrosis through the regulation of intestinal IgA+ immune cells.

Metabolic Profiling of Alpinetin in Rat Plasma, Urine, Bile and Feces after Intragastric Administration.

Alpinetin, a bioactive flavonoid, has been known to have a diverse therapeutic effect, with namely anti-inflammatory, anticancer and antioxidant effects with low systemic toxicity. This study aimed to obtain metabolic profiles of alpinetin in orally administrated rats. The metabolites of alpinetin were systematically analyzed and identified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The chromatographic separation was achieved on a High Strength Silica (HSS) T3 (1.8 µm, 2.1 × 100 mm) column with the mobile phase consisting of water containing 0.1% formic acid and acetonitrile with 0.1% formic acid via gradient elution. An extracted ion chromatogram strategy based on multiple prototype/metabolite intermediate templates and 71 typical metabolic reactions was proposed to comprehensively profile the metabolites of alpinetin. With the metabolite profiling strategy, altogether 15 compounds were recognized from urine, plasma, bile and feces of rats after intragastric administration of alpinetin for the first time. The prototype, glucuronide conjugates and phenolic acids metabolites were the probable predominant form of alpinetin in rats. This work showed a comprehensive study of the probable metabolic pathways of alpinetin in vivo, which could provide meaningful information for future pharmacological studies.

Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii.

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.

Peroxiredoxin 6 modulates Toll signaling pathway and protects DNA damage against oxidative stress in red swamp crayfish (Procambarus clarkii).

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.

Effective Protection on Acute Liver Injury by Halo Tag-Flanked Recombinant Fibroblast Growth Factor 7.

The drug development of FGF7 has been restricted by its toxicity to the host, low expression, poor stability, and easy degradation. Recent studies have shown that Halo-tag-flanked recombinant human FGF7 can solve the problem of toxicity; however, its biological activity is unknown. This study aimed to explore the activity of Halo-rhFGF7 and rhFGF7 on acute liver injury in vitro and in vivo. The rhFGF7 is expressed with a N-terminal Halo-tag, followed by a tobacco etch virus (TEV) protease cleavage site, in Escherichia coli BL21 (DE3) pLysS in this study. The products could stimulate the proliferation of carbon tetrachloride-damaged L-O2 cells (normal human liver cells); they also inhibited cell apoptosis. Due to the use of the Halo, the protein could be tracked using fluorescence localization. Recombinant protein exerted a protective effect on the acute liver injury model in vitro and in vivo. The MTT assay and Western blot analysis showed that this protective effect is realized through various paths, including promoting proliferation, inhibiting cell apoptosis and anti-inflammatory. In conclusion, Halo-rhFGF7 and rhFGF7 displayed an excellent protective effect on acute liver injury. The present study provided an experimental basis and data support for further research on rhFGF7.

Essential role of the peroxiredoxin 4 in Procambarus clarkii antioxidant defense and immune responses.

Peroxiredoxin (Prx) family members play a key role in host defense against oxidative stress, and modulate immune responses following microbial infection. Here, we cloned and characterized Procambarus clarkii Prx4 (Peroxiredoxin 4) cDNA, a regulator of oxidative stress and its expression analysis upon lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C) infection. The cDNA fragment of PcPrx4 was 744 bp in length, encoding a putative protein of 248 amino acid residues. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PcPrx4 was expressed in all the examined tissues, and it was highest in the hepatopancreas followed by the hemocytes and gill. The challenge with LPS and Poly I:C significantly up-regulated the expression of PcPrx4 in hepatopancreas, hemocytes and gill when compared with the control. Recombinant PcPrx4 protein was used to investigate the antioxidant function in vitro by mixed-function oxidase assay. The results demonstrated a dose-dependent inhibition of DNA damage by rPcPrx4 protein. Altogether, our results imply that PcPrx4 is implicated in defense against microbial pathogens and oxidants in P. clarkii.

Design, synthesis, and evaluation of NDGA analogues as potential anti-ischemic stroke agents.

Exogenous supplementation of antioxidants with ROS scavenging activity would be a potential therapy to cerebral ischemia-reperfusion injury in stroke. In the present study, a series of NDGA analogues with attenuation oxidative stress by directly scavenging ROS and indirectly through keap1/Nrf2/ARE pathway activation were designed and synthesized. All analogues were found to effectively remove ROS directly by DPPH radical scavenging assay, and compound 3a conferred potent protection from the oxidative injury in PC12 cells via promoting Nrf2 to translocate into nucleus and increasing expression of heme oxygenase-1(HO-1), where strongly reduced intracellular ROS level indirectly. More importantly, 3a significantly reduced brain infarction after cerebral ischemia-reperfusion injury in rats subjected to transient middle cerebral artery occlusion (MCAO). Overall, our findings shown compound 3a could serve as a promising compound for the treatment of stroke.

A role of cathepsin L gene in innate immune response of crayfish (Procambarus clarkii).

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In the present study, cathepsin L gene from the red crayfish Procambarus clarkii, named PcCTSL, was cloned and characterized. The cDNA fragment of PcCTSL was 1026 bp in length, which encoded a putative protein of 341 amino acid residues with a molecular weight of 37.884 kDa. The theoretical isoelectric point was 5.218. The prepro-cathepsin L was comprised of a typical signal peptide (Met1-Ala18), a prodomain proregion peptide (Trp29-Phe89) and a mature peptide (Leu124-Leu340). Homology analysis indicated that PcCTSL exhibited 53.2%-87.1% identity to other selected species. The recombinant protein of PcCTSL was successfully expressed in Escherichia coli and rabbit anti-PcCTSL polyclonal antibodies were prepared. Real-time quantitative reverse transcription-PCR (qPCR) analysis revealed that the PcCTSL was expressed in all examined tissues, while the greatest mRNA level was observed in hepatopancreas. The expression of PcCTSL mRNA was clearly up regulated in hepatopancreas after challenge by lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). RNA interference of PcCTSL affected the gene expression of members of the Toll pathway. Our results suggest that the PcCTSL may play an important role to defend P. clarkii against the pathogens infection.

Fibroblast growth factor 18 promotes proliferation and migration of H460 cells via the ERK and p38 signaling pathways.

Recently, fibroblast growth factor 18 (FGF18) expression was reported to be upregulated in colon cancer and ovarian cancer, and increased expression of FGF18 mRNA and protein is associated with tumor progression and poor overall survival in patients; however, its role in lung cancer remains to be explored. In the present study, the effect and underlying molecular mechanisms of FGF18 on H460 cells were investigated. Cell proliferation and cell cycle alterations were detected using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometry. A wound healing assay was conducted to detect cell migration. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to measure extracellular signal-regulated kinase (ERK), p38 and matrix metalloproteinase 26 (MMP26) expression. Knockdown of FGF18 using short interfering RNA (siRNA-FGF18) suppressed H460 cell proliferation, inhibited cell migration via the downregulation of MMP26 levels, with siRNA-FGF18 additionally inhibiting the ERK and p38 signaling pathway. The present study indicates that FGF18 serves an essential role in the growth and migration of non-small cell lung cancer (NSCLC) cells by regulating the ERK, p38 signaling pathways and MMP26 protein levels, suggesting that FGF18 may be a potential molecular drug target for the treatment NSCLC.

Application of oleosin-flanked keratinocyte growth factor-2 expressed from Arabidopsis thaliana promotes hair follicle growth in mice.

OBJECTIVE: To determine an efficient expression strategy in Arabidopsis thaliana and to determine whether a dimeric oleosin fusion approach could achieve keratinocyte growth factor-2 (KGF2) expression and bioactivity. RESULTS: Higher recombinant protein accumulation was observed in the two dimeric oleosin constructs than in a single-oleosin-fused protein (O::KGR2) or KGF2 control. Highest expression was in O-O::KGF2-transgenic seeds. MTT assay in FGFR2 III b-BaF3 cells revealed comparable levels of bioactivity due to O-O::KGF2 and O::KGR2, whereas O::KGF2-O had no effect on FGFR2 III b-BaF3 opithelial cell growth. The transgenic proteins had a pronounced stimulatory effect on hair follicle proliferation in C57BL/6 mice. CONCLUSION: A dimeric oleosin approach can be used to express KGF2 with the non-symmetrical O-O::KGF2 construct showing the highest expression and bioactivity.

Ginsenoside Rg5 improves cognitive dysfunction and beta-amyloid deposition in STZ-induced memory impaired rats via attenuating neuroinflammatory responses.

Neuroinflammatory responses play a crucial role in the pathogenesis of Alzheimer's disease (AD). Ginsenoside Rg5 (Rg5), an abundant natural compound in Panax ginseng, has been found to be beneficial in treating AD. In the present study, we demonstrated that Rg5 improved cognitive dysfunction and attenuated neuroinflammatory responses in streptozotocin (STZ)-induced memory impaired rats. Cognitive deficits were ameliorated with Rg5 (5, 10 and 20mg/kg) treatment in a dose-dependent manner together with decreased levels of inflammatory cytokines TNF-α and IL-1ß (P<0.05) in brains of STZ rats. Acetylcholinesterase (AChE) activity was also significantly reduced by Rg5 whereas choline acetyltransferase (ChAT) activity was remarkably increased in the cortex and hippocampus of STZ-induced AD rats (P<0.05). In addition, Congo red and immunohistochemistry staining results showed that Rg5 alleviated Aß deposition but enhanced the expressions of insulin-like growth factors 1 (IGF-1) and brain derived neurophic factor (BDNF) in the hippocampus and cerebral cortex (P<0.05). Western blot analysis also demonstrated that Rg5 increased remarkably BDNF and IGF-1 expressions whereas decreased significantly Aß deposits (P<0.05). Furthermore, it was observed that the expressions of COX-2 and iNOS were significantly up-regulated in STZ-induced AD rats and down-regulated strongly (P<0.05) by Rg5 compared with control rats. These data demonstrated that STZ-induced learning and memory impairments in rats could be improved by Rg5, which was associated with attenuating neuroinflammatory responses. Our findings suggested that Rg5 could be a beneficial agent for the treatment of AD.

[Effect of qingxin kaiqiao formula and saponin on learning and memory abilities and expression of apoptosis signal transducers Abeta and betaAPP in AD rat brain].

OBJECTIVE: To study the effect of qingxin kaiqiao formula and saponin on the learning and memory ability and the expression of the apoptosis signal transducers Abeta and betaAPP in AD rat brain. METHOD: The comparative observation method was adopted for the animal test. Forty male SD rats were randomly divided into five groups, namely the normal group, the model group, the aricept group, the qingxin kaiqiao formula group and the saponin group, with eight rats in each group. Abeta(25-35) (10 g x L(-1)) was injected into their bilateral amygdala to establish the AD rat model. Since the next day, they were intragastrically administered with Aricept (1.67 mg x kg(-1)), Qingxin Kaiqiao decoction (12.67 mL x kg(-1)), saponin (6.30 mg x kg(-1)) and double distilled water filling for 2 weeks to observe their spatial memory ability in a Morris water maze and study the expression of Caspase-3, Abeta and betaAPP in brain tissues by immunohistochemistry. RESULT: Each traditional Chinese medicine groups showed significant improvement in the learning and memory ability of AD rats and notable differences (P < 0.05, P < 0.01) compared with the control group. The qingxin kaiqiao formula group and the saponin group showed a decrease in the expressions of Caspase-3, Abeta and betaAPP in cerebral cortex and hippocampus area, displaying notable differences (P < 0.01, P < 0.05) compared with the control group. CONCLUSION: qingxin kaiqiao formula and saponin can obviously improve the learning and memory ability of AD rats with by decreasing the expression of Caspase-3, Abeta and betaAPP in cortex and hippocampus.

(E)-3-(2-Bromo-phen-yl)-1-(3,4-dimeth-oxy-phen-yl)prop-2-en-1-one.

The crystal structure of the title compound, C(17)H(15)BrO(3), a chalcone derivative, exhibits two crystallographically independent mol-ecules per asymmetric unit showing an E conformation about the ethyl-ene double bond. In each mol-ecule, the two phenyl rings are almost coplanar: the mean planes make dihedral angles of 9.3 (2) and 19.4 (2)°. In the crystal, mol-ecules are linked through weak inter-molecular C-H⋯O hydrogen bonds.