Molecular Cloning,Characterization and Expression Analysis of Woodchuck Retinoic Acid-Inducible GeneⅠ

Cytosolic retinoic acid-inducible gene I（RIG-I） is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β（IFN-β）. Innate immune response to hepatitis B virus（HBV） plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I（w RIG-I）, we analyzed the complete coding sequences（CDSs） of w RIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced w RIG-I protein was 106.847 k D with a theoretical isoelectric point（p I） of 6.07, and contained three important functional structures [caspase activation and recruitment domains（CARDs）, DEx D/H-box helicases, and a repressor domain（RD）]. In woodchuck fibroblastoma cell line（WH12/6）, w RIG-I-targeted small interfering RNA（si RNA） down-regulated RIG-I and its downstrean effector–IFN-β transcripts under RIG-I＇ ligand, 5＇-ppp double stranded RNA（ds RNA） stimulation. We also measured m RNA levels of w RIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus（WHV）-infected woodchucks. The basal expression levels of w RIG-I were abundant in the kidney and liver. Importantly, w RIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

Andrographolide as An Anti-H1N1 Drug and the Mechanism Related to Retinoic Acid-Inducible Gene-I-Like Receptors Signaling Pathway

Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Methods： Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line （16HBE）. The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium （MTT） assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma （IFN- ~, ） and interleukin-4 （IL-4） enzyme-linked immunosorbent assay （ELISA） detection while immunological cells for real-time polymerase chain reaction （RT-PCR）. Results： The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 （IPS-1）, interferon regulatory factor （IRF）-7, IRF-3 and nuclear transcription factor K B （NF- K B） mRNA levels increased significantly （P〈0.05）. When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences （P〈0.05）. Conclusions： The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes

AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Paramyxovirus evasion of innate immunity: Diverse strategies for common targets

The paramyxoviruses are a family of > 30 viruses that variously infect humans, other mammals and fish to cause diverse outcomes, ranging from asymptomatic to lethal disease, with the zoonotic paramyxoviruses Nipah and Hendra showing up to 70% case-fatality rate in humans. The capacity to evade host immunity is central to viral infection, and paramyxoviruses have evolved multiple strategies to overcome the host interferon(IFN)-mediated innate immune response through the activity of their IFN-antagonist proteins. Although paramyxovirus IFN antagonists generally target common factors of the IFN system, including melanoma differentiation associated factor 5, retinoic acid-inducible gene-I, signal transducers and activators of transcription(STAT)1 and STAT2, and IFN regulatory factor 3, the mechanisms of antagonism show remarkable diversity between different genera and even individual members of the same genus; the reasons for this diversity, however, are not currently understood. Here, we review the IFN antagonism strategies of paramyxoviruses, highlighting mechanistic differences observed between individual species and genera. We also discuss potential sources of this diversity, including biological differences in the host and/or tissue specificity of different paramyxoviruses, and potential effects of experimental approaches that have largely relied on in vitrosystems. Importantly, recent studies using recombinant virus systems and animal infection models are beginning to clarify the importance of certain mechanisms of IFN antagonism to in vivo infections, providing important indications not only of their critical importance to virulence, but also of their potential targeting for new therapeutic/vaccine approaches.

Differential roles of RIG-Ⅰ like receptors in SARS-CoV-2 infection

Retinoic acid-inducible gene Ⅰ (RIG-Ⅰ) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses.Herein we investigate their functions in human epithelial cells,the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).A deficiency in MDA5,RIG-Ⅰ or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication.The expression of the type I/III interferon(IFN) during infection was impaired in MDA5;and MAVS;,but not in RIG-Ⅰ;,when compared to wild type (WT) cells.The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2),the cellular entry receptor for SARS-CoV-2,was approximately 2.5-fold higher in RIG-Ⅰ;than WT cells.These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor,IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-Ⅰ in epithelial cells.

RIG-I,a novel DAMPs sensor for myoglobin,activates NF-κB/caspase-3 signaling in CS-AKI model

Background:Acute kidney injury(AKI)is the main life-threatening complication of crush syndrome(CS),and myoglobin is accepted as the main pathogenic factor.The pattern recognition receptor retinoicacid-inducible gene I(RIG-I)has been reported to exert anti-viral effects function in the innate immune response.However,it is not clear whether RIG-I plays a role in CS-AKI.The present research was carried out to explore the role of RIG-I in CS-AKI.Methods:Sprague-Dawley rats were randomly divided into two groups:the sham and CS groups(n=12).After administration of anesthesia,the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions.The rats in both groups were denied access to food and water.Rats were sacrificed at 12 h or 36 h after pressure was relieved.The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination.In addition,RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI.Furthermore,NRK-52 E cells were treated with 200μmol/L ferrous myoglobin to mimic CS-AKI at the cellular level.The cells and cell supernatant samples were collected at 6 h or 24 h.Small interfering RNAs(siRNA)was used to knock down RIG-I expression.The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative real-time PCR(qPCR),Western blotting analysis,and immunohistochemistry(IHC)staining.Tumor necrosis factor-α(TNF-α)was d etected by ELISA.Co-immunoprecipitation(Co-IP)assays were used to detect the interaction between RIG-I and myoglobin.Results:RNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway.qPCR,Western blotting,and IHC assays showed that RIG-I,nuclear factor kappa-B(NF-κB)P65,p-P65,and the a poptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group(P<0.05).However,the levels of interferon regulatory factor 3(IRF3),p-IRF3 and the antiviral factor interferon-beta(IFN-β)showed no significant c hanges between the sham and CS groups.Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group.Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis.C onclusions:RIG-I is a novel damage-associated molecular patterns(DAMPs)sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI.In the development of CS-AKI,specific intervention in the RIG-I p athway might be a potential therapeutic strategy for CS-AKI.

Effectiveness of a hydroxynaphthoquinone fraction from Arnebia euchroma in rats with experimental colitis

AIM:To evaluate the potential effectiveness of hydroxynaphthoquinone mixture(HM)in rats with 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:Colitis was induced by intracolonic administration of TNBS(80 mg/kg,dissolved in 50%ethanol).Rats were treated daily for 7 d with HM(2.5,5,10 mg/kg)and mesalazine 100 mg/kg 24 h after TNBS instillation.Disease progression was monitored daily by observation of clinical signs and body weight change.At the end of the experiment,macroscopic and histopathologic lesions of rats were scored,and myeloperoxidase(MPO)activity was determined.We also determined inflammatory cytokine tumor necrosis factor(TNF)-αlevel by ELISA,Western blotting and immunochemistry to explore the potential mechanisms of HM.RESULTS:After intracolonic instillation of TNBS,animals developed colitis associated with soft stool,diarrhea and marked colonic destruction.Administration of HM significantly attenuated clinical and histopathologic severity of TNBS-induced colitis in a dose-dependent manner.It abrogated body weight loss,diarrhea and inflammation,decreased macroscopic damage score,and improved histological signs,with a significant reduction of inflammatory infiltration,ulcer size and the severity of goblet cell depletion(all P<0.05 vs TNBS alone group).HM could reduce MPO activity.In addition,it also decreased serum TNF-αlevel and down-regulated TNF-αexpression in colonic tissue.This reduction was statistically significant when the dose of HM was 10 mg/kg(P<0.05 vs TNBS alone group),and the effect was comparable to that of mesalazine and showed no apparent adverse effect.The underlying mechanism may be associated with TNF-αinhibition.CONCLUSION:These findings suggest that HM possesses favourable therapeutic action in TNBS-induced colitis,which provides direct pharmacological evidence for its clinical application.

Pharmacokinetic Evaluation of Chitosan-Succinyl-Prednisolone Conjugate Microparticles as a Colonic Delivery System: Comparison with Enteric-Coated Conjugate Microparticles

In the previous study, chitosan-succinyl-prednisolone conjugate microparticles (MP) were found to exhibit good efficacy and reduced toxicity nearly as well as their Eudragit L-coated microparticles (MP/EuL). This proposes a question whether enteric-coating of MP is necessary or not. Although MP/EuL were already examined for their pharmacokinetic and gastrointestinal behaviors, MP have not been done yet. Therefore, in this study, MP were evaluated by investigating pharmacokinetic features in detail. MP with the in vitro features equivalent to those of the previous conjugate microparticles could be produced more readily in the modified preparative method. Pharmacokinetic and gastrointestinal behaviors of MP were investigated by intragastric dosing (5 mg PD eq./kg) to rats with 2,4,6-trinitrobenzenesulfonic acid-induced ulcerative colitis. The plasma concentration was suppressed extensively in MP as well as MP/EuL, supporting the reduction of PD systemic toxic side effects. However, the plasma level increased gradually up to 7 h in MP, but not in MP/EuL. At 8 h after dosing, MP were detected in the stomach to a fair extent, and free PD was found there, indicating that MP were subjected to trapping in the stomach probably due to positive charge of chitosan molecules. For MP, such prolonged localization and slow release of PD in the stomach were probably associated with the gradual increase in plasma concentration. Therefore, MP/EuL were evaluated to be superior to MP for effective targeting to ulcerative colitis. It is concluded that enteric-coating is very important for the targeting system using MP.

Therapeutic and Immunoregulatory Effect of GATA-Binding Protein-3/T-Box Expressed in T-Cells Ratio of Astragalus Polysaccharides on 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis in Rats

Objective： To analyze the immunological characteristics of 2,4,6-trinitrobenzene sulfonic acid（TNBS）-induced colitis model and examine the therapeutic effects and mechanisms of Astragalus polysaccharides（APS） treatment. Methods： Thirty-two male specific pathogen free Spragne-Dawley rats were randomly equally assigned to four groups： control, TNBS, APS and prednisone groups. Experimental colitis was induced by enema administration of TNBS. Then rats were treated with APS（0.5 g·kg^（-1）·day^（-1）, once daily） or prednisone（1.0 mg·kg^（-1）·day^（-1）, once daily） by gavage for 14 days. Macroscopic lesion and histological damage were determined, and activity of myeloperoxidase（MPO） was measured in the colonic tissues. Expressions of T-box expressed in T-cells（T-bet） and GATA-binding protein-3（GATA-3） were determined by immunohistochemistry analysis and western blot. Results： Both macroscopic lesion and histological colonic damage induced by TNBS were reduced by APS and prednisone treatment. These were accompanied by significant attenuation of MPO activity（P=0.03）. TNBS intervention enhanced the expression of both GATA-3 and T-bet, but the expression of T-bet was significantly enhanced than that of GATA-3, resulting in significant reduction of GATA-3/T-bet ratio（P=0.025）. APS administration enhanced the expression of T-bet（P=0.04） and GATA-3（P=0.019） in comparison to TNBS group, and resulting in an up-regulated GATA-3/T-bet ratio. Prednisone treatment inhibited both expressions; however it also resulted in up-regulation of the GATA-3/T-bet ratio. Conclusions： These results demonstrated that APS exerted a beneficial immune regulatory effect on experimental colitis. It promoted the expression of T helper cell 1（Th1） and T helper cell 2（Th2） specific transcription factors but ultimately favor a shift toward Th2 phenotype, suggesting that APS possessed therapeutic potential in experimental colitis.

Healing effects of heparin on acetic acid-induced gastric ulcers in rats

Abstract Objective To investigate whether or not heparin can accelerate the healing process of acetic acid induced gastric ulcers in rats and to identify the mechanisms for heparin to produce this effect, so that we can develop a new therapeutic application to heparin besides its traditional anticoagulant activity. Methods Male Sprague Dawley rats were used to produce acetic acid induced gastric ulcers. Heparin in the doses of 100, 500, and 1000 U/kg were administered intravenously through the tail vein once daily, starting 1 day after ulcer induction for 7 days in the dose response experiment or heparin 1000 U/kg at a time schedule of 3, 5, and 7 days in the time response study, respectively. The gastric mucosal blood flow (GMBF) was measured using a laser Doppler flowmeter under ether anesthesia. The rats were then sacrificed and the ulcer areas were measured. The gastric mucosa was then scraped for the determinations of mucosal prostaglandin E 2 (PGE 2) level and myeloperoxidase (MPO) activity. Results Heparin in the doses of 500 and 1000 U/kg accelerated the healing of acetic acid ulcers in a dose dependent manner. The highest dose of heparin also reduced the ulcer areas in a time dependent fashion. The effect was accompanied by an increase in gastric mucosal PGE 2 levels. The same dose of heparin not only decreased the gastric mucosal MPO activity but also increased the GMBF in a time related manner. Conclusions Heparin with the doses used in the present study accelerated the healing of acetic acid induced gastric ulcers in rats in a dose and time dependent manner, and this action was related to its effects to increase the levels of gastric mucosal PGE 2 and GMBF as well as to decrease the gastric mucosal MPO activity.

Acid-induced topological morphology modulation of graphitic carbon nitride homojunctions as advanced metal-free catalysts for OER and pollutant degradation

Topological morphology that dominates the surface electronic properties of nanostructures plays a key role in producing desired materials for versatile functions and applications in many fields,but its modulation for specific functions remains a big challenge.Herein,we report an acid-induced method to prepare S-doped graphitic carbon nitride/graphitic carbon nitride(S-CN/CN)homojunction by simply pyrolyzing a supramolecular precursor synthesized from melamine and H_(2)SO_(4).The topological morphology and electronic structure of CN homojunction can be easily adjusted only by changing the ratio of raw materials.Moreover,the topological morphology of S-CN/CN homojunction can be further adjusted from hollow cocoon to 2D nanosheets by changing the annealing conditions.The optimized S-CN/CN homojunction shows highly efficient in charge transfer and separation and exhibits superior OER activity and high ability to degrade organic pollutants.Impressively,S-CN/CN nanosheets only demand low overpotential of301 m V to drive a current density of 10 m Acm^(-2)in 1 M KOH media,and the corresponding Tafel slope is only 57.71 m V/dec,which is superior to the most advanced precious metal Ir O_(2)catalyst.Moreover,under visible light irradiation,its photodegradation kinetic rate of Rh B is 2.38,which is 47.6 times higher than that of bulk CN.This work provides useful guidance for designing and developing efficient multifunctional metal-free catalysts.

Three dimensional phytic acid-induced graphene as a solid-phase microextraction fiber coating and its analytical applications for nerolidol in tea

In this work, three dimensional phytic acid-induced graphene coating was prepared by hydrothermal synthesis and fabricated on a stainless-steel wire using physical coating method as a solid phase microextraction （SPME） coating. The graphene coating revealed high specific surface area, extraordinary electrical conductivity and excellent chemical stability. Coupled with gas chromatography-flame ionization detector （GC-FID）, the home-made phytic acid-induced graphene SPME fiber exhibited excellent extraction efficiency for the analysis of nerolidol in tea samples. Under the optimized conditions, the linear range of working curve was found to be 1-1000 ng/g, and the limit of detection of nerolidol was 0.18 ng/g. The coefficient variation of repeatability for each single fiber and reproducibility for fiber to fiber was less than 3.9Yg and 8.3~, respectively.

Rheological properties of peanut protein isolate aggregation suspension and acid-induced gel

The dynamic rheological properties of peanut protein isolate(PPI)suspension and acid-induced PPI gels were studied.In frequency sweep test,the storage modulus(G′)and the loss modulus(G″)of PPI aggregation suspensions at different concentrations increased with the increase of frequency.The steady state shear flow test showed that PPI aggregation suspension had a thinning behavior of the shear,and the image of steady shear curve fitted the Carreau model.After gel formation,acid-induced PPI gels showed a typical Type I behavior(strain thinning)in strain sweep test,meaning that PPI gel got easily broken down,and there was a very small opportunity for the protein molecules to re-establish the network.Compared with the strain sweep of PPI aggregation suspensions and gels,the range of the storage modulus existed a dramatic difference,which could get about tenfold.As the frequency increased,both elasticity and viscosity increased in frequency sweep test,which indicated that the frequency dependence of the storage modulus increased with the increase of concentration.

Endoplasmic Reticulum Stress Mediates Palmitic Acid-induced Insulin Resistance in Mouse Skeletal Muscle Cells

Increasing evidence shows that pathological elevation of plasma fatty acids, especially long-chain saturated forms, which ordinarily occurs in obesity patients, increases the risk

Effect of different forms of selenium in osteoporosis rat model induced by retinoic acid

Osteoporosis is a typical physiological disease,the main symptoms of which are brittle fracture,bone pain and easily deformed.As an individual ages,the prevalence of osteoporosis increases year by year.In the present study,selenium with antioxidant,immunomodulatory and anti-tumor effects was used to prevent osteoporosis induced by retinoic acid.The serum calcium contents in the selenium-treated groups(sodium selenite and selenomethionine)were significantly higher(P<0.05)than those in the model group in both the prevention and treatment studies.After pre-vention,glutamic-oxalacetic transaminase transaminase(GOT),glutamate transaminase(GPT),alkaline phosphatase(ALP)and tartrate-resistant acid phosphatase(TRACP)levels were significantly(P<0.05)decreased.In the treatment study,the serum calcium and phosphorus contents of the rats increased after selenium treatment.There was no significant change(P>0.05)in the activity of GOT and GPT.The content of ALP decreased obviously and the TRACP enzyme activity increased.Overall,these results showed that different forms of selenium compounds have great potential in preventing and treating osteoporosis.