Deciphering the molecular mechanisms of Simiaowan in the treatment of hyperuricemia: in vivo and in silico approaches

Background:Simiaowan(SMW),a well-known traditional Chinese medicine,has been employed to treat hyperuricemia(HUA)and gout for centuries.However,the bioactive components and underlying mechanisms have not been elucidated.The objective of this study was to identify the active components and potential mechanisms of SMW by integrating pharmacological experimentation,serum pharmacochemistry,network pharmacology and molecular docking.Methods:HUA rats modelling by high-fat/high-sugar diet and potassium oxonate/adenine oral administration were used to evaluate the pharmacodynamic effects of SMW.UPLC-Q-Exactive-MS/MS was employed to detect the bioactive components present in SMW-containing serum.Network pharmacology and molecular docking were utilized to elucidate the potential targets and underlying mechanisms.Results:SMW effectively ameliorated HUA rats via the inhibition of uric acid(UA)production,promotion of UA excretion,improvement of lipid and glucose metabolic abnormalities,antioxidant,anti-inflammatory and anti-insulin resistance effects.A total of 73 compounds detected in SMW-containing serum were identified as potential active components,with alkaloids,flavonoids,organic acids,and terpenoids emerging as the primary active ingredients.Totally 203 corresponding targets were obtained as SMW anti-HUA/gout targets,which mainly participated in apoptosis,insulin resistance,TNF,PI3K-Akt,HIF-1,NF-κB,MAPK,IL-17 and TLR signaling pathways.Molecular docking indicated that active compounds(e.g.berberine,phellodendrine,quercetin,formononetin,ferulic acid)had superior binding abilities to the key targets(e.g.solute carrier family 22 member 12(URAT1),solute carrier family 22 member 6(OAT1),ATP-binding cassette sub-family G member 2(ABCG2),solute carrier family 2,facilitated glucose transporter member 9(GLUT9),xanthine dehydrogenase/oxidase(XDH),transcription factor p65(RELA),toll-like receptor 4(TLR4),prostaglandin G/H synthase 2(PTGS2),caspase-3(CASP3),insulin(INS)).Conclusion:SMW exerted regulatory influence over the disease network of HUA and gout through a multiplicity of components,targets,and pathways.Alkaloids,flavonoids,organic acids,and terpenoids were the primary active components,exerting anti-HUA/gout effects via antioxidant,anti-inflammatory,anti-insulin resistance,anti-apoptosis,inhibition of UA production,and promotion of UA excretion.This study revealed the active components and molecular mechanisms of SMW,providing insights into the development of natural products derived from SMW.

Improved reversed phase liquid chromatographic method with pulsed electrochemical detection for tobramycin in bulk and pharmaceutical formulation

Tobramycin is one of the aminoglycoside antibiotics that lack a UV absorbing chromophore. However, the application of pulsed electrochemical detection （PED） has been used successfully for the analysis of this and similar antibiotics. This work describes an improved liquid chromatographic （LC） method combined with PED, which is able to separate much more impurities than before. Using a Discovery C-18 RP column （250 mm × 4.6 mm i.d., 5 μm）, isocratic elution was carried out with a mobile phase, containing sodium sulfate （35 g/L）, sodium octanesulphonic acid （1 g/L）, tetrahydrofuran （14mL/L） and 0.2 M phosphate buffer pH 3.0 （50 mL/L）. Using these experimental conditions, the limit of quantification （LOQ, S/N= 10） was 5 ng. The linearity was examined in the range LOQ-60 μg/mL and the coefficient of determination was 0.998. The method also proved to be repeatable and the recovery was close to 100%. The influence of the different chromatographic parameters on the separation was investigated by means of an experimental design. The proposed method is useful in quality control of tobramycin drug substances and drug products.

基于《江苏省医疗机构药品遴选与临床应用评价体系》的氟维司群的临床综合评价研究

目的:对新型内分泌治疗药物氟维司群进行药品评价,为医疗机构遴选该药品提供参考。方法:参考药品说明书、临床指南、相关文献、权威数据平台和网站等,使用《江苏省医疗机构药品遴选与临床应用评价体系》的评价维度、评价细则及评价方法,对氟维司群进行药品量化评分与评价。结果:基于评价体系的氟维司群总评分为55分:其中,必要性8分,安全性6分,有效性20分,经济性5分,创新性3分,适宜性4分,可及性4分,其他情况5分。结论:本研究中所用方法可供医疗机构的药物遴选与评价参考,能够帮助医疗卫生机构快速地评价与遴选药品,可为临床用药提供参考。

Pathogenic genes associated with Parkinson’s disease:molecular mechanism overview

Parkinson’s disease(PD)is a common neurodegenerative disease in the elderly,accounting for more than 1%of the population aged 65 years.Monogenic inheritance is relatively rare in PD,accounting for approximately 5%to 10%of PD patients,and there is a growing body of evidence suggesting that multiple genetic risk factors play a significant role in the pathogenesis of PD.Several groups have identified and reported a number of genes carrying mutations associated with affected family members.Mutated genes associated with PD are also candidates for idiopathic PD,and these genes may also carry other mutation sites that increase risk.When multiple genetic risk factors are combined,the risk of PD is increased to a greater extent,and to unravel the pathogenic pathways that lead to different forms of PD.This review focuses on the association of PD genes,such as Parkinson Disease 1-24(PARK1-24),glucosylceramidase(GBA),GTP cyclohydrolase 1(GCH1),fibroblast growth factor 20(FGF20),nuclear receptor-related factor 1(NURR1),NUS1 dehydrodolichyl diphosphate synthase subunit(NUS1),diacylglycerol Lipase Beta(DAGLB),transmembrane protein(TMEM),ubiquinol-cytochrome c reductase core protein 1(UQCRC1),glycoprotein non-metastatic melanoma protein B protein(GPNMB),dynactin 1(DCTN1),LDL receptor related protein 10(LRP10),monoamine oxidase(MAO),ataxin 2(ATXN2),microtubule associated protein tau(MAPT),pantothenate kinase 2(PANK2),spastic parapplegia type 11(SPG11),polymer gamma(POLG),TATA-box binding protein associated factor 1(TAF1),dual specificity tyrosine phosphorylation regulated kinase 1A(Dyrk1a),and crystallin alpha A(CRYAA),with the pathogenesis of PD.We introduce what is currently known about the molecular genetics of PD to help explain the molecular mechanisms leading to the neurodegenerative disease.

Lentivirus Transduced T Cell Lines Response to Pro-Inflammatory and Antiviral Cytokines in the Presence of HIV

Various studies have attempted to understand HIV infection under a diverse range of stimulants including cytokine stimulation. Pro-inflammatory cytokines, such as TNF-α, have been shown to reactivate HIV latency by inducing NF-κB mediated activation of the HIV LTR (long terminal repeats) that contain κB transcriptional binding sites. Interferon-alpha (IFN-α), an anti-viral cytokine, is not well studied as an inducer of HIV activation. However, previous work from our group has shown that HIV can block IFN-α signaling in CD4+ T cells presumably to allow for further viral replication. Initially using HEK 293T cells, we moved to CD4+ T cells lines to develop a system to determine how stimulation with different cytokines impacts signaling within T cell lines. We confirmed that in our system TNF-α triggers activation of NF-κB driven reporters but not in the presence of HIV. In addition, we show that the presence of HIV blocks IFN-α signaling. Taken together, our system demonstrates that HIV by TNF-α, will continue to block IFN-α signaling preventing it from impacting HIV activation. This system can now be used to screen for cytokine based and other molecule activators that may be influenced by the presence of HIV.

Neuroprotective effects of resveratrol on retinal ganglion cells in glaucoma in rodents:A narrative review

Glaucoma,an irreversible optic neuropathy,primarily affects retinal ganglion cells(RGC)and causes vision loss and blindness.The damage to RGCs in glaucoma occurs by various mechanisms,including elevated intraocular pressure,oxidative stress,inflammation,and other neurodegenerative processes.As the disease progresses,the loss of RGCs leads to vision loss.Therefore,protecting RGCs from damage and promoting their survival are important goals in managing glaucoma.In this regard,resveratrol(RES),a polyphenolic phytoalexin,exerts antioxidant effects and slows down the evolution and progression of glaucoma.The present review shows that RES plays a protective role in RGCs in cases of ischemic injury and hypoxia as well as in ErbB2 protein expression in the retina.Additionally,RES plays protective roles in RGCs by promoting cell growth,reducing apoptosis,and decreasing oxidative stress in H_(2)O_(2)-exposed RGCs.RES was also found to inhibit oxidative stress damage in RGCs and suppress the activation of mitogen-activated protein kinase signaling pathways.RES could alleviate retinal function impairment by suppressing the hypoxia-i nducible factor-1 alpha/vascular endothelial growth factor and p38/p53 axes while stimulating the PI3K/Akt pathway.Therefore,RES might exert potential therapeutic effects for managing glaucoma by protecting RGCs from damage and promoting their survival.

Effect of DC-CIK cell on the proliferation,apoptosis and differentiation of leukemia cells

Objective:To observe the effect of co-culture cytokine-induced killer cells(CIK) and homologous dendritic cells(DC) on the proliferative activity and phenotype change of the DCCIK cell and the cell killing activity of leukemia HL-60.Methods:50 mL cord blood sample was obtained from infants delivered by full term healthy woman and the cord blood mononuclear cells were isolated by density gradient centrifugal ion.Non-adherent cells were collectedfor the induction culture of CIK.adherent cells were differentiated into mature DC;cultured mature DC was mixed with and CIK in the proportion of 1:5 for 12 d.killing activity of DC-CIK co-cultured cell on leukemia HL-60 was detected by MTT assay.Results:Compared with CIKs.the cocultured DC-CIKs presented a markedly higher proliferation and killing activity.Conclusions:Co-culture of DC-CIK cells led to a significant increase of the proliferation and cytotoxicity of CIK.

Pulmonary delivery of liposomal dry powder inhaler formulation for effective treatment of idiopathic pulmonary fibrosis

Dry powder inhaler Liposomes were prepared to investigate the effectiveness of pulmonary delivery of Colchicine and Budesonide for Idiopathic Pulmonary fibrosis. Budesonide(BUD) and Colchicine(COL) liposomes were prepared by thin layer film hydration method(TFH) using 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol sodium(DPPG), Hydrogenated Soyaphosphotidylcholine(HSPC), Soyaphosphatidylcholine(SPC), cholesterol(CHOL) and drug in different weight ratios. The optimum lipid composition for BUD(74.22 ± 0.97%) was DPPG:HSPC: CHOL(4:5:1) and for COL(50.94 ± 2.04%) was DPPG: SPC: CHOL(3:6:1). These compositions retained drug for a longer period of time so selected for further study. Liposomes were found to be spherical in shape with mean size below 100 nm. Liposomes lyophilized using Mannitol as carrier and cryoprotectant showed high entrapment efficiency(97.89-98.6%). The powder was dispersed through an Andersen cascade impactor to evaluate the performance of the aerosolized powder. It was found that prepared liposomal dry powder inhaler(DPIs) sustained the drug release up to 24 hours. Optimized Budesonide DPI Formulation B2(86.53 ± 1.9%), Colchicine DPI Formulation C2(90.54 ± 2.3 %) and BUD and COL DPI Combination M2(89.91 ± 1.8%, 91.23 ± 1.9%). Histopathological results, measurements of lung hydroxyproline content, Myeloperoxidase activity indicated that liposomal drypowder inhaler administration attenuates lung fibrosis induced by bleomycin. Long term stability studies indicated that lyophilised BUD and COL liposomes were stable for 6 months at(25 °C± 2 °C, 60% ± 5% RH) and refrigerated conditions(2-8 °C). These results supported that combination of budesonide and colchicine liposomal dry powder inhaler pulmonary drug delivery for treatment of idiopathic Pulmonary Fibrosis exhibits prolonged drug retention at targeted site and reduces the systemic exposure.

Investigation of in vitro antioxidant activity of dihydromyricetin and flavonoids rich extract from vine tea(Ampelopsis grossedentata)

Background:Vine tea from fermented Ampelopsis grossedentata leaves has been used as a herbal tea and folk medicine in the southern region of China for hundreds of years.The aim of this investigation was to analyze the total flavonoids found in vine tea,including three bioactive flavonoids,and the total phenolic contents in the aqueous methanol extracts of 10 vine tea samples.In addition,this study also aimed to examine the antioxidant activity of dihydromyricetin and vine tea’s flavonoid-rich extract.Methods:The total flavonoids and total phenolic content assay of extracts from vine tea were performed by ultraviolet-visible spectroscopy and epoch microplate spectrophotometer,respectively.Three bioactive flavonoids were quantified simultaneously using high performance liquid chromatography.The antioxidant activity of dihydromyricetin and vine tea’s flavonoid-rich extract was evaluated in vitro using six different methods.Results:Vine tea contained a large number of flavonoids,with dihydromyricetin as its main constituent.The flavonoid-rich extract exhibited a significant scavenging effect on superoxide anion radicals,and on 3-ethylbenzthiazoline-6-sulphonic acid and 1,1-diphenyl-2-picrylhydrazyl radicals.It also possessed definite activity in lipid peroxidation inhibition,ferric reduction,and the moderation of Fe2+ion chelation ability.There was a significant negative correlation between dihydromyricetin content and antioxidant activity in the vine tea samples,including superoxide anion radical scavenging activity(P=−0.754,P<0.05),lipid peroxidation inhibition activity(P=−0.759,P<0.05),ferric-reducing antioxidant power(P=−0.843,P<0.01),respectively.Dihydromyricetin played a dominant role in the antioxidant activities of the flavonoid-rich extract.Conclusion:Vine tea’s flavonoid-rich extract could be used as a new antioxidant source to safeguard against oxidative stress.

Pulsatile core-in-cup valsartan tablet formulations:In vitro evaluation

The aim of the present study was to design and evaluate single pulse and floating double pulse valsartan core-in-cup tablets.Core tablets were prepared by direct compression of a homogenous mixture of valsartan,Avicel PH-101,Croscarmellose sodium(CCNa),magnesium stearate&Aerosil.Weight variation,Hardness and Disintegration time were measured for the core tablets.Core-in-cup tablets were formulated using different polymers as a plug layer,including sodium alginate(SA),sodium carboxymethylcellulose(NaCMC)and hydroxypropyl methyl cellulose(HPMC).The floating behavior,water uptake and drug release from the prepared formulations were evaluated.Differential Scanning Calorimetry(DSC)was also performed to detect the possible drug excipient interaction.Stability study of the selected formula was performed at 25
C&60%RH and at 40
C&75%RH for 3 months.Finally,the existence of the selected formula in the stomach after oral administration to human volunteers was verified via x-ray radiography.The results showed that the release lag time of the tablets increased when the quantity of the plug layer increased thus decreasing the drug release.Plug layer polymers showed a lag time with rank order:SA<NaCMC<HPMC.Selected formulations are F5&F6.F5(having SA as the plug polymer)released valsartan after a lag time of 2 h while F6 released the drug in two successive pulses with a reasonable lag time in between due to its floating behavior.Formulations were stable for at least 3 months under standard long-term and accelerated storage conditions.In conclusion,pulsatile single pulse and floating double pulse stable valsartan core-incup tablets were successfully formulated which provided a desirable lag time followed by a rapid drug release.

Genotypic Characterization of Methicillin-resistant Staphylococcus aureus Isolated from Pigs and Retail Foods in China

Objective To investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus （MRSA） isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food. Methods Seventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing （MLST）, spa typing, multiple-locus variable number of tandem repeat analysis （MLVA）, pulsed-field gel electrophoresis （PFGE）, and antimicrobial susceptibility testing. Results All isolated MRSA exhibited multi-drug resistance （MDR）. Greater diversity was found in food-associated MRSA （7 STs, 8 spa types, and 10 MLVA patterns） compared to pig-associated MRSA （3 STs, 1 spa type, and 6 MLVA patterns）. PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates （40 vs. 11 pulse types）. Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone （96.4%）, and CC9-ST9-t437-MC621 （20.0%） was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains （t030, t172, t1244, and t4549） were also identified as food-associated isolates. Conclusion CC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.

Repositioning of dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 agonists as potential neuroprotective agents

Repositioning of dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists is a breakthrough in the field of neural regeneration research increasing glucagon like peptide-1 bioavailability, hence its neuroprotective activities. In this article, the authors suggest not only crossing blood-brain barrier and neurodegenerative disease as off target for dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists, but also for ophthalmic preparations for diabetic retinopathy, which may be the latest breakthrough in the field if prepared and used in an appropriate nano-formulation to target the retinal nerves. The relation of neurodegenerative diseases' different mechanisms to the dipeptidyl peptidase-4 inhibitors and glucagon like peptide-1 receptor agonists should be further examined in preclinical and clinical settings. The repositioning of already marketed antidiabetic drugs for neurodegenerative diseases should save the high cost of the time-consuming normal drug development process. Drug repositioning is a hot topic as an alternative to molecular target based drug discovery or therapeutic switching. It is a relatively inexpensive pathway due to availability of previous pharmacological and safety data. The glucagon like peptide-1 produced in brain has been linked to enhanced learning and memory functions as a physiologic regulator in central nervous system by restoring insulin signaling. Intranasal administration of all marketed gliptins(or glucagon like peptide-1 receptor agonists) may show enhanced blood-brain barrier crossing and increased glucagon like peptide-1 levels in the brain after direct crossing of the drug for the olfactory region, targeting the cerebrospinal fluid. Further blood-brain barrier crossing tests may extend dipeptidyl peptidase-4 inhibitors' effects beyond the anti-hyperglycemic control to intranasal spray, intranasal powder, or drops targeting the blood-brain barrier and neurodegenerative diseases with the most suitable formula. Moreover, novel nano-formulation is encouraged either to obtain favorable pharmacokinetic parameters or to achieve promising blood-brain barrier penetration directly through the olfactory region. Many surfactants should be investigated either as a solubilizing agent for hydrophobic drugs or as penetration enhancers. Different formulae based on in vitro and in vivo characterizations, working on sister gliptins(or glucagon like peptide-1 receptor agonists), different routes of administration, pharmacokinetic studies, dose response relationship studies, monitoring of plasma/brain concentration ratio after single and multiple dose, and neurodegenerative disease animal models are required to prove the new method of use(utility) for dipeptidyl peptidase-4 inhibitors as potential neuroprotective agents. Furthermore, investigations of glucagon like peptide-1 receptor agonists' neuroprotective effects on animal models will be considered carefully because they crossed the blood-brain barrier in previous studies, enabling their direct action on the central nervous system. Combination therapy of dipeptidyl peptidase-4 inhibitors or glucagon like peptide-1 receptor agonists with already marketed drugs for neurodegenerative disease should be considered, especially regarding the novel intranasal route of administration.

新型液相色谱-荧光检测法测定药物剂型和人血浆中的他达拉非和盐酸达泊西汀(英文)

Tadalafil(TAD)and dapoxetine HCl(DAP)are recently co-formulated and both show native fluorescence.Therefore,a novel,accurate,specific and sensitive reversed-phase high performance liquid chromatographic method with fluorescence detection was developed and validated for their separation and quantitation in dosage form and human plasma using avanafil as an internal standard(IS).Separation was achieved using isocratic elution within 7.0 min on C18column and acetonitrile-0.15%triethylamine(40∶60,v/v;pH 4)as a mobile phase.The flow rate was 1.0 mL/min and the detection was time-programmed at 330,410 and 370 nm for TAD,DAP and IS,respectively,after excitation at 236 nm.The linear ranges from 0.01 to 30.00μg/mL for each drug with the limits of detection of 4.20 and 7.20 ng/mL for TAD and DAP,respectively.The method was validated in accordance to the International Conference on Harmonization(ICH)guidelines and was successfully applied to spiked human plasma with mean recoveries of 98.17%and 98.83%for TAD and DAP respectively.

Commentary of the mRNA vaccines COVID-19

The World Health Organization(WHO)declared global SARS-CoV-2(COVID-19)pandemic status on March 11,2020[1].To date,data record approximately 106 million infected individuals and 2.32 million deaths due to pandemic COVID-19,worldwide.The most common symptoms of SARS-CoV-2 infection reported are:elevation of body temperature,fatigue,cough,loss of smell.In a percentage of patients,especially elderly individuals with comorbidities,COVID-19 infection can cause severe organ injury[2].Since the onset of the COVID-19 pandemic,numerous pharmacological treatments have been used off label,to treat the viral infection,with the primary aim of avoiding the most serious complications and organ injury.

Influence of bioactive sulphated polysaccharide-protein complexes on hepatocarcinogenesis, angiogenesis and immunomodulatory activities

Objective:To explore the in vivo anticancer,anti-angiogenesis and immunomodulatory efficacies of the bioactive polysaccharide isolated from cold aqueous extract of Jania rubens(JCEM) and Pterocladia capillacea(PCEM) as well as hot aqueous extract of Enteromorpha intestinalis(EHEM) against hepatocellular carcinoma rat model(HCC) and to study their chemical composition.Methods:The sugars and amino acids composition of the bioactive polysaccharides of JCEM,PCEM and EHEM were determined using gas liquid chromatography and amino acid analyzer,respectively.These polysaccharide extracts(20 mg/kg b.wt.for 5 weeks) were assessed on hepatocarcinogenesis in rats and α-fetoprotein(AFP),carcinoembryonic antigen(CEA),glypican-3(GPC-3),hepatocyte growth factor(HGF) and vascular endothelial growth factor(VEGF) and Ig G levels were evaluated.Results:The GLC analysis of JCEM,PCEM and EHEM polysaccharide revealed the presence of 10,9 and10 sugars,in addition the amino acid analyser enable identification of 16,15 and 15 amino acids,respectively.These polysaccharide extracts of JCEM,PCEM and EHEM produced significant decrease in serum AFP,CEA,GPC-3,HGF and VEGF compared with untreated HCC group.JCEM,PCEM and EHEM had an immunostimulatory responses by increasing the IgG levels as compared by naive value(1.23,1.53 and 1.17 folds),respectively.The bioactive polysaccharides in HCC induced rats improved the humoral immune response.The photomicrographs of liver tissue sections of the groups of HCC treated with polysaccharide extracts of Jania rubens and Enteromorpha intestinalis showed intact histological structure.Moreover,fractions HE1,HE4,HE7 obtained from polysaccharide of EHEM showed moderate cytotoxic activity against Hep G2 in vitro with IC_(50) 73.1,42.6,76.2 μg/mL.However,fractions of PCEM and JCEM show no or weak cytotoxicity against Hep G2 in vitro where the cytotoxic activity of their crude polysaccharide extract proved synergetic effect.Conclusions:The pronounced antitumor activity of sulphated polysaccharide-protein complexes of JCEM and EHEM is due to direct cytotoxic activity,anti-hepatocarcinogensis,and anti-angiogenesis.In addition,JCEM,PCEM and EHEM had an immunostimulatory response and improved the humoral immune response in HCC induced rats.

Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients

AIM: To investigate the relationship of changes in expression of marker genes in functional categories or molecular networks comprising one functional category or multiple categories in progression of hepatic fibrosis in hepatitis C (HCV) patients. METHODS: Marker genes were initially identified using DNA microarray data from a rat liver fibrosis model. The expression level of each fibrosis associated marker gene was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in clinical biopsy specimens from HCV-positive patients (n = 61). Analysis of changes in expression patterns and interactions of marker genes in functional categories was used to assess the biological mechanism of fibrosis. RESULTS: The profile data showed several biological changes associated with progression of hepatic fibrosis. Clustered genes in functional categories showed sequential changes in expression. Several sets of clustered genes, including those related to the extracellular matrix (ECM), inflammation, lipid metabolism, steroid metabolism, and some transcription factors important for hepatic biology showed expression changes in the immediate early phase (F1/F2) of fibrosis. Genes associated with aromatic amino acid (AA) metabolism, sulfur-containing AA metabolism and insulin/ Wnt signaling showed expression changes in the middle phase (F2/F3), and some genes related to glucose metabolism showed altered expression in the late phase of fibrosis (F3/F4). Therefore, molecular networks showing serial changes in gene expression are present in liver fibrosis progression in hepatitis C patients. CONCLUSION: Analysis of gene expression profiles from a perspective of functional categories or molecular networks provides an understanding of disease and suggests new diagnostic methods. Selected marker genes have potential utility for biological identification of advanced fibrosis.

A stepwise optimization strategy to formulate in situ gelling formulations comprising fluconazole-hydroxypropyl-beta-cyclodextrin complex loaded niosomal vesicles and Eudragit nanoparticles for enhanced antifungal activity and prolonged ocular delivery

Fungal keratitis and endopthalmitis are serious eye diseases.Fluconazole(FL)is indicated for their treatment,but suffers from poor topical ocular availability.This study was intended to improve and prolong its ocular availability.FL niosomal vesicles were prepared using span 60.Also,polymeric nanoparticles were prepared using cationic Eudragit RS100 and Eudragit RL100.The investigated particles had adequate entrapment efficiency(EE%),nanoscale particle size and high zeta potential.Subsequently,formulations were optimized using full factorial design.FL-HP-β-CD complex was encapsulated in selected Eudragit nanoprticles(FL-CD-ERS1)and niosmal vesicles.The niosomes were further coated with cationic and bioadhesive chitosan(FL-CD-Nios-ch).EE%for FL-CD-ERS1 and FL-CD-Niosch formulations were 76.4%and 61.7%;particle sizes were 151.1 and 392 nm;also,they exhibited satisfactory zeta potential+40.1 and+28.5 m V.In situ gels were prepared by poloxamer P407,HPMC and chitosan and evaluated for gelling capacity,rheological behavior and gelling temperature.To increase the precorneal residence time,free drug and selected nano-formulations were incorporated in the selected in situ gel.Release study revealed sustained release within 24 h.Permeation through excised rabbits corneas demonstrated enhanced drug flux and large AUC0-6 h in comparison to plain drug.Corneal permeation of selected formulations labeled with Rhodamine B was visualized by Confocal laser microscopy.Histopathological study and in vivo tolerance test evidenced safety.In vivo susceptibility test using Candida albicans depicted enhanced growth inhibition and sustained effect.In this study the adopted stepwise optimization strategy combined cylodextrin complexation,drug nano-encapsulation and loading within thermosenstive in situ gel.Finally,the developed innovated formulations displayed boosted corneal permeation,enhanced antifungal activity and prolonged action.

Peroxynitrite-induced expression of inducible nitric oxide synthase and activated apoptosis via nuclear factor-kappa B pathway in retinal pigment epithelial cells and antagonism of cholecystokinin octapeptide-8 in vitro

AIM: To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase (iNOS)via nuclear factor-kappa B (NF-kappa B)pathway in retinal pigment epithelial (RPE) cells and the antagonism of cholecystokinin octapeptide-8 (Melatonin, CCK-8) in vitro. METHODS: RPE cells were obtained from eyes of C57BL/6 mouse and divided into control, peroxynitrite and CCK-8 groups. Control group was treated with saline, peroxynitrite group was treated with peroxynitrite, and CCK-8 group was treated with CCK-8 after added with peroxynitrite. All changes were observered at 6, 12 and 24 hours after treatment. Gene array analysis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to determine the expression of inducible nitric oxide synthase ( iNOS)mRNA in RPE cells. Western blotting was used to test the apoptosis of RPE cells. Immunofluorescent staining was used to determine the NF-kappa B pathway signal transduction. RESULTS: Compared to the control group, the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis. Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting. The NF-kappa B pathway signal transduction was more and more stronger in the peroxynitrite group. But in CCK-8 group, little stronger could be observed at 12 hours, then weak at 24 hours with immunofluorescent staining (P<0.001). CONCLUSION: This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite, which may be the new way of oxidative damage to the RPE cells. The NF-kappa B signal transduction may affect and reinforce apoptosis mediated by peroxynitrite. CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.

Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

AIM: To determine the gene expression profile data for the whole liver during development of dimethylni-trosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells), and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells. RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSC-specific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis, suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocyte-specific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis. CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibro-genesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

Pivotal role of long non-coding ribonucleic acid-X-inactive specific transcript in regulating immune checkpoint programmed death ligand 1 through a shared pathway between miR-194-5p and miR-155-5p in hepatocellular carcinoma

BACKGROUND Anti-programmed death therapy has thrust immunotherapy into the spotlight.However,such therapy has a modest response in hepatocellular carcinoma(HCC).Epigenetic immunomodulation is a suggestive combinatorial therapy with immune checkpoint blockade.Non-coding ribonucleic acid(ncRNA)driven regulation is a major mechanism of epigenetic modulation.Given the wide range of ncRNAs that co-opt in programmed cell-death protein 1(PD-1)/programmed death ligand 1(PD-L1)regulation,and based on the literature,we hypothesized that miR-155-5p,miR-194-5p and long non-coding RNAs(lncRNAs)X-inactive specific transcript(XIST)and MALAT-1 are involved in a regulatory upstream pathway for PD-1/PD-L1.Recently,nutraceutical therapeutics in cancers have received increasing attention.Thus,it is interesting to study the impact of oleuropein on the respective study key players.AIM To explore potential upstream regulatory ncRNAs for the immune checkpoint PD-1/PD-L1.METHODS Bioinformatics tools including microrna.org and lnCeDB software were adopted to detect targeting of miR-155-5p,miR-194-5p and lncRNAs XIST and MALAT-1 to PD-L1 mRNA,respectively.In addition,Diana tool was used to predict targeting of both aforementioned miRNAs to lncRNAs XIST and MALAT-1.HCC and normal tissue samples were collected for scanning of PD-L1,XIST and MALAT-1 expression.To study the interaction among miR-155-5p,miR-194-5p,lncRNAs XIST and MALAT-1,as well as PD-L1 mRNA,a series of transfections of the Huh-7 cell line was carried out.RESULTS Bioinformatics software predicted that miR-155-5p and miR-194-5p can target PDL1,MALAT-1 and XIST.MALAT-1 and XIST were predicted to target PD-L1 mRNA.PD-L1 and XIST were significantly upregulated in 23 HCC biopsies compared to healthy controls;however,MALAT-1 was barely detected.MiR-194 induced expression elevated the expression of PD-L1,XIST and MALAT-1.However,overexpression of miR-155-5p induced the upregulation of PD-L1 and XIST,while it had a negative impact on MALAT-1 expression.Knockdown of XIST did have an impact on PD-L1 expression;however,following knockdown of the negative regulator of X-inactive specific transcript(TSIX),PD-L1 expression was elevated,and abolished MALAT-1 activity.Upon co-transfection of miR-194-5p with siMALAT-1,PD-L1 expression was elevated.Co-transfection of miR-194-5p with siXIST did not have an impact on PD-L1 expression.Upon co-transfection of miR-194 with siTSIX,PD-L1 expression was upregulated.Interestingly,the same PD-L1 expression pattern was observed following miR-155-5p cotransfections.Oleuropein treatment of Huh-7 cells reduced the expression profile of PD-L1,XIST,and miR-155-5p,upregulated the expression of miR-194-5p and had no significant impact on the MALAT-1 expression profile.CONCLUSION This study reported a novel finding revealing that opposing acting miRNAs in HCC,have the same impact on PD-1/PD-L1 immune checkpoint by sharing a common signaling pathway.