Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

Fenofibrate Pre-treatment Suppressed Inflammation by Activating Phosphoinositide 3 Kinase/Protein Kinase B(PI3K/Akt) Signaling in Renal Ischemia-Reperfusion Injury

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury（IRI） in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups： sham-operated group（sham）, IRI＋saline group（IRI group）, IRI＋Fenofibrate（FEN） group. Normal saline or Fenofibrate（3 mg/kg） was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8（IL-8）, tumor necrosis factor alpha（TNF-α） and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B（PI3K/Akt） signaling and peroxisome proliferator-activated receptor-α（PPAR-α） were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

HSP110 aggravates ischemia-reperfusion injury after liver transplantation by promoting NF-κB pathway

Background:Ischemia-reperfusion injury(IRI)poses a significant challenge to liver transplantation(LT).The underlying mechanism primarily involves overactivation of the immune system.Heat shock protein 110(HSP110)functions as a molecular chaperone that helps stabilize protein structures.Methods:An IRI model was established by performing LT on Sprague-Dawley rats,and HSP110 was silenced using siRNA.Hematoxylin-eosin staining,TUNEL,immunohistochemistry,ELISA and liver enzyme analysis were performed to assess IRI following LT.Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes.Results:Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT(P<0.05).However,when rats were injected with siRNAHSP110,IRI subsequent to LT was notably reduced(P<0.05).Additionally,the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced(P<0.05).Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver(P<0.05).Conclusions:HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells.Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI.

Bioactive chemical constituents from the marine-derived fungus Cladosporium sp.DLT-5

A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidation determined the planar structure of 1.Subsequent electronic circular dichroism(ECD)experiment assigned the absolute configuration of 1.Compounds 1,2,4–6,and 10 displayed different degrees of neuroprotective activities on human neuroblastoma cells SH-SY5Y.Five compounds(1,3–5,and 13)emerged resistance to protein tyrosine phosphatase 1B(PTP1B),further kinetic analysis and molecular docking study indicated that the most potent compound 13(IC50value of 10.74±0.61μmol/L)was found as a noncompetitive inhibitor for PTP1B.Surface plasmon resonance(SPR)and molecular docking studies also demonstrated the interaction between compound 12 and Niemann-Pick C1 Like 1(NPC1L1),which has been identified as significant therapeutic target for hypercholesteremia.In addition,compounds 3,6,and 14 showed attractive inhibitory activity against the phytopathogenic fungi:Colletotrichum capsici.Therefore,library of Cladosporium metabolites is enriched and new active uses of known compounds are explored.

Promising Effects of Zerumbone on the Regulation of Tumor-promoting Cytokines Induced by TNF-α-activated Fibroblasts

Inflammation plays an important role in the development of several cancers.Inflammatory cytokines,including tumor necrosis factor-α(TNF-α),are associated with the induction of inflammation.Chronic inflammation contributes to the progression of cancer through several mechanisms,including increased cytokine production and activation of transcription factors,such as nuclear factor-κB(NF-κB).Zerumbone(ZER),a component of subtropical ginger(Zingiber zerumbet Smith),seems to have anti-inflammatory,anti-cancer,and antioxidant activities.In this study,we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines.We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER(IC50=18µmol/L),compared to un-stimulated fibroblasts(IC50=40µmol/L).Besides,ZER inhibited mRNA expression and protein secretion of transforming growth factor-β(TGF-β),interleukin-33(IL-33),monocyte chemoattractant protein-1(MCP-1),and stromal cell-derived factor 1(SDF-1),which were produced by TNF-α-induced fibroblasts,as measured by quantitative real time-PCR(qRT-PCR)and ELISA assays.The mRNA expression levels of TGF-β,IL-33,SDF-1,and MCP-1 showed 8,5,2.5,and 4-fold reductions,respectively.Moreover,secretion of TGF-β,IL-33,SDF-1,and MCP-1 was reduced to 3.65±0.34 ng/mL,6.3±0.26,1703.6±295.2,and 5.02±0.18 pg/mL,respectively,compared to the untreated group.In addition,the conditioned media(CM)of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines(HCT-116 and Sw48),while in the vicinity of ZER,the expression of NF-κB was reversed.Considering the significant effects of ZER,this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

灵芝提取物通过PBX3/MAPK通路对胶质瘤细胞恶性生物学行为的作用机制研究

目的探究灵芝提取物(GLE)是否可通过前B细胞白血病同源盒基因3(PBX3)/丝裂原活化蛋白激酶(MAPK)通路影响U251人胶质瘤细胞恶性生物学行为。方法2022年1月至2023年1月进行该研究。含不同浓度GLE培养液(0、50、100、200 mg/L GLE)培养U251细胞48 h,采用细胞计数试剂盒(CCK-8)法、流式细胞术、平板克隆实验、细胞划痕试验及迁移和侵袭(Transwell)实验来评估细胞的存活率、凋亡情况、集落形成能力、迁移与侵袭特性;实时定量聚合酶链反应(RT-qPCR)检测PBX3、细胞外信号调节酶(ERK)mRNA表达水平;蛋白质印迹法检测PBX3、原癌基因c-RAF(Raf-1)、磷酸化Raf-1(p-Raf-1)、信号通路细胞外信号调节酶1/2(ERK1/2)、磷酸化ERK1/2(p-ERK1/2)蛋白表达情况。结果0、50、100、200 mg/L GLE下U251细胞存活率分别为100%、(86.62±4.26)%、(67.68±3.49)%、(50.84±3.39)%、(40.13±3.25)%,差异有统计学意义(P<0.05);0、50、100、200 mg/L GLE下U251细胞凋亡率、集落形成数、划痕愈合率、侵袭细胞数、PBX3、ERK mRNA及PBX3、p-Raf-1、p-ERK1/2蛋白相对表达水平比较,差异有统计学意义(P<0.05);随着GLE浓度的增加,U251细胞存活率、划痕愈合率、PBX3与ERK mRNA相对表达水平及RAS、PBX3、p-Raf-1、p-MEK1/2、p-ERK1/2蛋白相对表达水平均降低,集落形成数及侵袭细胞数均减少,细胞凋亡率升高;GLE作用效果呈剂量性依赖(P<0.05)。结论GLE可抑制胶质瘤细胞增殖、克隆形成、迁移及侵袭等恶性生物学特性,并诱导其凋亡,其作用机制可能与阻断PBX3/MAPK通路的激活相关。

PM_(2.5) obtained from urban areas in Beijing induces apoptosis by activating nuclear factor-kappa B

Background: Particulate matter(PM), which has adverse effects on citizen health, is a major air pollutant in Beijing city. PM_(2.5) is an indicator of PM in urban areas and can cause serious damage to human health. Many epidemiological studies have shown that nuclear factor-kappa B(NF-κB) is involved in PM_(2.5)-induced cell injury, but the exact mechanisms are not well understood.Methods: The cytotoxic effects of PM_(2.5) at 25–1600μg/ml for 24 h were determined by MTT assay in Chinese hamster ovary cells(CHO) cells. Flow cytometry was used to determine the apoptosis rate induced by PM_(2.5). The destabilized enhanced green fluorescent protein(d2 EGFP) green fluorescent protein reporter system was used to determine the NF-κB activity induced by PM_(2.5). The expression of pro-apoptotic Bcl-2-associated death promoter(BAD) proteins induced by PM_(2.5) was determined by Western blotting to explore the relationship between PM_(2.5) and the NF-κB signaling pathway and to determine the toxicological mechanisms of PM_(2.5).Results: PM_(2.5) collected in Beijing urban districts induces cytotoxic effects in CHO cells according to MTT assay with 72.28% cell viability rates even at 200μg/ml PM_(2.5) and flow cytometry assays with 26.97% apoptosis rates at 200μg/ml PM_(2.5). PM_(2.5) increases the activation levels of NF-κB, which have maintained for 24 h. 200μg/ml PM_(2.5) cause activation of NF-κB after exposure for 4 h, the activation peak appears after 13.5 h with a peak value of 25.41%. The average percentage of NF-κB activation in whole 24 h is up to 12.90% by 200μg/ml PM_(2.5). In addition, PM_(2.5) decreases the expression level of the pro-apoptotic protein BAD in a concentration-dependent manner.Conclusion: PM_(2.5) induces NF-κB activation, which persists for 24 h. The expression of pro-apoptotic protein BAD decreased with increased concentrations of PM_(2.5). These findings suggest that PM_(2.5) plays a major role in apoptosis by activating the NF-κB signaling pathway and reducing BAD protein expression.

Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

Recent studies have shown that varied stress stimuli activate c-Jun N-terminal kinase （JNK）, protein kinase B （Akt）, and p38 mitogen-activated protein kinase （p38） signal transduction pathway, and also regulate various apoptotic cascades. JNK and p38 promote apoptosis, but Akt protects against apoptosis, in hippocampal neurons. However, changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood. Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group （P 〈 0.05）. No significant difference in JNK phosphorylation levels was detected in the rat cerebral cortex between stress and control groups, and no significant difference in Akt and p38 phosphorylation levels was detected in the rat hippocampus and cerebral cortex between stress and control groups （P 〉 0.05）. These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

miR-126调控MAPK/NF-κB信号通路对急性肺损伤大鼠的干预效果

目的 分析微小RNA-126(microRNA-126,miR-126)通过调控丝裂原活化蛋白激酶(MAPK)/核转录因子κB(NF-κB)信号通路对急性肺损伤大鼠的干预效果。方法 选取50只Wistar大鼠,10只作为对照组,不做任何处理,其余40只建立急性肺损伤模型,将建模成功30只大鼠分为模型组、miR-126沉默组、miR-126过表达组,每组10只。观察各组大鼠一般情况、病理组织学、miR-126表达量、氧化应激指标、血气分析指标、炎性因子、p38 MAPK、NF-κB p65 mRNA信号通路相关mRNA表达量、MAPK/NF-κB信号通路。结果 与对照组比较,模型组肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、p38 MAPK mRNA、NF-κB p65 mRNA、p38 MAPK、NF-κB p65升高,miR-126、超氧化物歧化酶(SOD)活性、血氧分压(PaO_(2))降低(P<0.05);与模型组比较,miR-126沉默组TNF-α、p38 MAPK mRNA、NF-κB p65 mRNA、p38 MAPK、NF-κB p65升高,miR-126、SOD活性、PaO_(2)降低,miR-126过表达组TNF-α、IL-1β、p38 MAPK mRNA、NF-κB p65 mRNA、p38 MAPK、NF-κB p65降低,miR-126、SOD活性、PaO_(2)升高(P<0.05)。结论 过表达miR-126可抑制MAPK/NF-κB信号通路的表达,降低炎性反应,改善大鼠氧化应激指标、血气分析指标及病理学变化。

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-κB activation

AIM： To study the effects of hepatitis C virus （HCV） core and non-structural 5A （NS5A） proteins on nuclear factor- k B （NF- k B） activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS： Luciferase assay was used to measure the activity of NF-kB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF- k B-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF- k B/p65, NF- k B/p50, and inhibitor k B-a（k B-a）. RESULTS： The wild-type core protein （C191） and its mutant segments （C173 and C158） could activate NF- k B in Huh7 cells only and activation caused by （C191） could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF- k B. The NF- k B activity was augmented due to the dissociation of NF-k： B-I k： B complex and the degradation of Ik B-a. CONCLUSION：NF- k B is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF- k B activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.

Donkey whey proteins ameliorate dextran sulfate sodium-induced ulcerative colitis in mice by downregulating the S100A8-TRAF6-NF-κB axis-mediated inflammatory response

Donkey milk has a variety of physiological functions, including antibacterial and anti-inflammatory. Donkey whey proteins(DWPs), as the main functional component in donkey milk, its inhibitory effect on colitis is still unclear. In this study, the inhibitory effect and potential mechanism of DWPs on dextran sulfate sodium(DSS)-induced colitis were investigated. Firstly, the DWPs and bovine milk whey proteins(BWPs)were characterized using proteomics. Then, we administered DWPs and BWPs to mice with colitis via oral gavage. The results of immunohistochemistry and flow cytometry indicated that DWPs increased T regulatory cell accumulation and increased the abundance of the cluster of differentiation 205+(CD205+)macrophages compared to those with BWPs and in model groups. In addition, DWPs exhibited a more remarkable ability to inhibit pro-inflammatory proteins(S100A8, TRAF6, and NF-κB)expression and inflammatory secretion than BWPs. In addition, DWPs significantly decreased NF-κB and CD86 levels more than BWPs or the negative control in both LPS-stimulated human peripheral blood mononuclear cells or cell lines. These findings indicate that DWPs comprise a promising anti-colitis functional food, and this work has established a foundation for future research on these compounds.

Relationship between Carbachol Hyperstimulation-induced Pancre-atic Intracelluar Trypsinogen and NF-κB Activation in Rats in vitro

The relationship between intracelluar trypsinogen activation and NF-κB activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist （carbachol） hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc） and NF-κB inhibitor （PDTC） in vitro. Intracelluar trypsin activity was measured by using a fluorogenic substrate. The activity of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-κB in pancreatic acinar cells treated with high concertrations of carbachol （10^-3 mol/L） in vitro was significantly decreased as compared with control group （P〈0.01 ）. The addition of 10^-2 mol/L PDTC resulted in a significant decrease of NF-κB activities in pancreatic acinar cells after treated with high concertrations of carbachol （10^-3 mol/L） in vitro, but the intracelluar trypsinogen activity was not obviously inhibited （P〉0.05）. It was concluded that intracelluar trypsinogen activation is likely involved in the regulation of high concertrations of carbachol-induced NF-κB activation in pancreatic acinar cells in vitro. NF-κB activation is likely not necessary for high concertrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Relationship between Carbachol Hyperstimulation-Induced Pancreatic Acinar Cellular Injury and Trypsinogen or NF-κB Activation in Rats in vitro

The relationship between M3 cholinergic receptor agonist （carbachol） hyperstimulationinduced pancreatic acinar cellular injury and trypsinogen activation or NF-κB activation in rats was studied in vitro. Rat pancreatic acinar ceils were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc）, and NF-κB inhibitor （PDTC） in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar ceils. The results showed that as compared with control group, 10-3 mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells （P〈0. 01） following the treatment with a high concentration of carbachol （10^-3 mol/L） in vitro. The addition of 10^-2mol/L PDTC didn＇t result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol （10^-3 mol/L） in vitro （P〉0. 05）. It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-κB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Rhamnus crenata leaf extracts exhibit anti-inflammatory activity via modulating the Nrf2/HO-1 and NF-κB/MAPK signaling pathways

Objective:To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells.Methods:We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability.Nitric oxide(NO)production was measured using Griess reagent.Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators,respectively.Moreover,PD98059(ERK1/2 inhibitor),SB203580(p38 inhibitor),SP600125(JNK inhibitor),and BAY11-7082(NF-κB inhibitor)were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract.Results:Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO,iNOS,COX-2,IL-1β,and TNF-αin lipopolysaccharide(LPS)-stimulated RAW264.7 cells.Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-αand nuclear accumulation of p65,which resulted in the inhibition of NF-κB activation in RAW264.7 cells.Additionally,the extracts attenuated the phosphorylation of p38,ERK1/2,and JNK in LPS-stimulated RAW264.7 cells.Moreover,HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580,PD98059,SP600125 and BAY11-7082.Conclusions:Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38,ERK1/2,and NF-κB activation,which may contribute to the anti-inflammatory activity of the extracts.Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

Effects of Echinacea Polysaccharide on Expression of NF-κB Protein Secreted by LPS-injured IEC-6 Cells

[ Objective ] The paper was to explore effects of Echinacea polysaccharide （EPS） on expression of NF-KB protein secreted by LPS-injured IEC-6 cells, and to provide a theoretical basis for clinical application of Echinacea purpurea against bacterial diseases and enhancement of immunity. [ Method] Nucleoprotein extracted from IEC-6 cells in normal control group, LPS group, different concentrations of EPS （50, 100,200,500 μg/mL） ＋ LPS groups were detected by SDS- PAGE electrophoresis, and the content of NF-κB protein was analyzed using western blotting method. [ Result ] The content of NF-KB protein in normal control group was the lowest, while that in LPS group was the highest. The content of NF-κB protein in EPS group gradually decreased with the increasing concentration of EPS. [ Result] The expression of NF-κB protein increased when IEC-6 cells were stimulated by LPS, and EPS could effectively inhibit increased expression of NF- κB protein. With the increasing concentration of EPS, the inhibition effect against increased expression of NF-κB protein gradually strengthened.

Inhibitory Actions of Tetrandrine on Tumor Necrosis Factor α-Induced NF-κB Activation in Neovascularization of Cultured Choroidal Explants

Tetrandrine (1 μM), a bis-benzylisoquinoline alkaloid isolated from Stephania tetrandra S Moore, signifi-cantly decreased tumor necrosis factor alpha (TNFα;10 ng/ml)-induced increase in the number of micro vessels that budded from cultured rat choroidal explants. Tetrandrine also decreased the TNFα-induced in-crease in the number of cells composing the microvessels. Ammonium pyrrolidine dithiocarbamate (APDC;0.1-0.3 μM), an inhibitor of nuclear factor-κB (NF-κB), decreased the TNFα-induced increase in the number of microvessels in a concentration-dependent manner. TNFα increased the phosphorylation and degradation of inhibitor of NF-κB (IκBα), as well as increasing the DNA-binding activity of NF-κB in choroidal explants. TNF? induced an increase of vascular endothelial growth factor (VEGF)-A mRNA, but not VEGF-C mRNA or VEGF-D mRNA. TNFα-induced angiogenic action was inhibited by treatment of VEGF-A antibody in cultured choroidal capillaries. Tetrandrine inhibited the TNFα-induced increases of phosphorylation and degradation of IκBα, and reduced the TNFα-induced increase of DNA-binding activity of NF-κB in chor-oidal explants. In conclusion, tetrandrine inhibits TNFα-induced activation of NF-κB in the choroidal capil-laries via inhibition of TNFα-induced phosphorylation of IκBα.

Inhibitory roles of protein kinase B and peroxisome proliferator-activated receptor gamma coactivator on hepatic HMG-CoA reductase promoter activity

Since we had previously demonstrated that siRNAs to tristetraprolin (TTP) markedly inhibited insulin stimulation of hepatic HMG-CoA reductase (HMGR) transcription, we investigated the effects of transfecting rat liver with TTP constructs. We found that transfecting diabetic rats with TTP did not increase HMGR transcription but rather led to modest inhibition. We then investigated whether co-transfection with protein kinase B, hepatic form (AKT2), might lead to phosphorylation and result in activation of HMGR transcription. We found that this treatment resulted in near complete inhibition of transcription. Transfection with peroxisome proliferator-activated receptor g coactivator (PGC-1a) also inhibited HMGR transcription. These results show that although TTP is needed for activation of HMGR transcription, it cannot by itself activate this process. AKT2 and PGC-1a, which mediate the activation of gluconeogenic genes by insulin, exert the opposite effect on HMGR.

STAT3、ERK、NF-κB在黄芪保护病毒感染心肌细胞中的作用

目的:研究STAT3、ERK、NF-κB在黄芪保护感染病毒的心肌细胞中的作用。方法:体外分离培养心肌细胞并分为对照组;病毒感染组:病毒孵育;黄芪干预组:病毒孵育后分别给予黄芪培养浓度为300 mg/L、500mg/L、700 mg/L。采用RT-PCR检测各组病毒RNA,采用W esten b lotting检测P-STAT3、P-ERK、NF-κB p65表达。MTT法观察细胞活性。结果:在各组中STAT3、ERK总蛋白没有明显变化(P>0.05),病毒组P-STAT3、P-ERK、NF-κB p65明显高于对照组(P<0.01),黄芪组P-STAT3、P-ERK、NF-κB p65明显低于病毒组(P<0.01)。黄芪组细胞存活率明显高于病毒组(P<0.01)。结论:在病毒性心肌炎中黄芪可以通过抑制STAT3、ERK、NF-κB保护心肌细胞。