Knockdown of STAT3 by iRNA Inhibiting Migration and Invasion of Epithelial Ovarian Cancer Cells

Signal transducer and activator of transcription 3（STAT3） is a dual functional transcription factor with the functions of signal transduction and transcription regulation. It is reported that the expression of STAT3 in ovarian cancer is significantly higher and STAT3 can facilitate ovarian cancer growth and metastasis. To clarify the definite effect and molecular mechanism of STAT3 involved in ovarian cancer growth and metastasis, STAT3 expression was significantly downregulated by transfecting ovarian cancer model SK-OV-3 cells with the plasmid vector which express specific RNAi that targets human STAT3. The downregulated STAT3 not only decreased the invasion and migration but also inhibited the proliferation of SK-OV-3 cells. Western blot assay shows that the expression of vascular endothelial growth factor（VEGF） and that of Survivin were reduced in the cells with the plasma vector expressing specific RNAi that targets human STAT3. These results demonstrate that STAT3 involved in the invasion and migration of SK-OV-3 regulates the expression of VEGF and Survivin. In addition, VEGF and Survivin could play an important role in ovarian cancer growth and metastasis.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

Objective:This study explores the mechanism of action of Danhongqing formula(DHQ),a compoundbased Chinese medicine formula,in the treatment of cholestatic liver fibrosis.Methods:In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout(Mdr2-/-)mice as an animal model of cholestatic liver fibrosis.DHQ was administered orally for 8 weeks,and its impact on cholestatic liver fibrosis was evaluated by assessing liver function,liver histopathology,and the expression of liver fibrosis-related proteins.Real-time polymerase chain reaction,Western blot,immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19(H19)and signal transducer and activator of transcription 3(STAT3)phosphorylation in the liver tissue of Mdr2-/-mice.In addition,cholangiocytes and hepatic stellate cells(HSCs)were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression.Cholangiocytes overexpressing H19 were constructed,and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation.The intervention effect of DHQ on these processes was also investigated.HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ.Results:DHQ alleviated liver injury,ductular reaction,and fibrosis in Mdr2-/-mice,and inhibited H19expression,STAT3 expression and STAT3 phosphorylation.This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19,inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium,and decreased the expression of activation markers in HSCs.The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation,and DHQ was able to successfully inhibit these effects.Conclusion:DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/-mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC,thereby suppressing cell activation.

Jianpi Qutan Fang(健脾祛痰方)induces anti-atherosclerosis and ameliorates endothelial cell injury in high-fat diet rats via an anti-inflammatory and inhibiting Janus kinase/signal transducer and activator of transcription signaling pathway

OBJECTIVE:To investiage the possible mechanism underlying the effect of the Jianpi Qutan Fang(健脾祛痰方,JPQT)on Atherosclerosis(AS)which is the main pathological process of most cardiovascular diseases that affect millions of adults worldwide.METHODS:In the present study,rats were fed with a high-fat-diet(HFD)with vitamin D3 for 16 weeks and were orally administered atorvastatin treatment and different doses of JPQT.Histopathological changes and ultrastructural changes in the aorta were evaluated through hematoxylin-eosin staining and transmission electron microscopy(TEM),respectively.Suppressor of cytokine signaling 1(SOCS1)/Janus kinase 1(JAK1)/signal transducer and activator of transcription 1(STAT1)signaling pathways were detected through Western blotting.RESULTS:JPQT treatment decreased the lipid levels of triglyceride,low-density lipoprotein,and cholesterol,the inflammatory cytokine levels of interleukin 1 beta(IL-1β),IL-6 and IL-8 in rat serum,but increased high-density lipoprotein and IL-10 serum levels.JPQT treatment ameliorated pathological changes in the aorta of AS model rats.Moreover,JPQT upregulated SOCS1 protein expression and down-regulated phosphorylated protein expression levels of p-JAK1 and p-STAT1.CONCLUSION:These results suggest that JPQT induces anti-atherosclerosis effects through anti-inflammatory and inhibiting JAK/STAT signaling pathways in HFD fed rats.

Protective effect of Chushizi(Fructus Broussonetiae)on acetaminophen-induced rat hepatitis by inhibiting the Toll-like receptor 3/c-Jun N-terminal kinase/c-jun/c-fos/janus protein tyrosine kinase/activators of transcription 3 pathway

OBJECTIVE:To observe the intervention of Chushizi(Fructus Broussonetiae)(CSZ)on drug-induced liver injury(DILI)in rats,as well as indicators of liver function,serum levels of inflammatory cytokines,and expression of proteins and m RNA associated with toll-like receptor 3(TLR3)and the signal transducer and activator of transcription 3(STAT3)pathway in the liver[TLR3,janus protein tyrosine kinase 2(JAK2),c-jun,c-fos,c-Jun N-terminal kinase 2(JNK2),and STAT3].METHODS:Forty specified pathogen free grade Sprague-Dawley rats were randomly divided into the control group,the model group,the silybin group and the CSZ group.Rats were given acetaminophen(APAP)to trigger DILI.Histopathology of the liver was observed by hematoxylin-eosin staining.The levels of alanine aminotransferase(ALT),aspartate transaminase(AST),direct bilirubin(DBIL),and total bilirubin(TBIL)in serum were detected by a semi-automatic biochemical instrument.Content of tumor necrosis factor alpha(TNF-α),interleukin(IL)-6,IL-13,and IL-22 in serum were detected by the enzyme-linked immunosorbent assay,the expression of TLR3,phosphorylation of JAK2(p-JAK2),while c-jun and c-fos proteins in the liver were determined by immunohistochemistry;expression of JNK2,and STAT3 in the liver were assayed by Western blot and real-time quantitative polymerase chain reaction.P-JNK2 and p-STAT3 in the liver were assayed by Western blot.RESULTS:After treatment,the activity of ALT,AST,and concentrations of TBIL,DBIL,TNF-α,IL-6,as well as IL-13 in serum,were lower than those in the model group,and expression of p-JAK2,TLR3,c-jun,c-fos,p-STAT3,and p-JNK2 could be downregulated.CONCLUSION:Our findings suggest that CSZ is a valid medicine to alleviate APAP-induced DILI,while its partial mechanism may regulate the TLR3/JNK/c-jun/c-fos/JAK/STAT3 pathway.

Effect of RNA Interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions

Background： Signal transducer and activator of transcription 3 （STAT3） was strongly expressed and activated in psoriatic keratinocytes （KCs） and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA （siRNA） combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods： Psoriatic KCs were transfected under four experimental conditions： （1） STA T3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （LUS group）; （2） STA T3 siRNA only carried by Lipofectamine 3000 （L group）; （3） the negative control ofsiRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （siRNA-NC）; （4） not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration （[Ca^2＋）. The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction （RT-PCR） and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results： STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. ST,4 T3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STAT3 siRNA induced the elevation of [Ca^2＋] of KCs with the highest calcium fluorescence intensity mean of 1213.67 ±60.51 in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STA T3 siRNA induced the downregulation ofcyclin D 1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58%±4.92% and 64.06% ±7.78% at mRNA level and at protein level, respectively （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. Conclusions： STA T3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca^2＋], and downregulating cyclin DI and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.

Effects of peroxisome proliferator-activated receptor-β/δ on sepsis induced acute lung injury

Background Acute lung injury （ALI） and acute respiratory distress syndrome （ARDS） are the first steps in the development of multiple organ failure induced by sepsis.A systemic excessive inflammatory reaction is currently the accepted mechanism of the pathogenesis of sepsis.Several studies have suggested a protective role of the peroxisome proliferator activated receptor-β/δ （PPAR-β/δ） in related inflammatory diseases.But the role of PPARβ/δ in ALI remains uncertain.The aim of this study was to investigate the role and possible mechanism of PPARβ/δ in ALI induced by sepsis.Methods Cecal ligation and puncture （CLP） was used as a sepsis model.Rats were randomly divided into four groups,the control group （CON,n=6）,sham-operation group （SHAM,n=12）,cecal ligation and puncture group （CLP,n=30）,GW501516 group （CLP＋GW,n=25）,which underwent CLP and were subcutaneously injected with the PPAR-β/δ agonist GW501516 （0.05 mg/100 g body weight）.Survival was monitored to 24 hours after operation.Blood pressure,serum creatinine,blood urea nitrogen,aspartate aminotrasferase and alanine aminotrasferase were measured after CLP.Concentrations of tumor necrosis factor α （TNF-α） and interleukin （IL）-1β in serum were detected by enzyme linked immunosorbent assay （ELISA） kits.Lung tissue samples were stained with H＆E and scored according to the degree of inflammation.Bacterial colonies were counted in the peritoneal fluid.Alveolar macrophages were cultured and incubated with GW501516 （0.15 μmol/L） and PPARβ/δ adenovirus and then treated with Lipopolysaccharide （2 μg/ml） for 2 hours.The TNF-α,IL-1β and IL-6 RNA in lung and alveolar macrophages were determined by real-time PCR.Phosphorylation of signal transducer and activator of transcription 3 （STAT3） in lung and alveolar macrophages was detected by Western blotting.Results GW501516 significantly increased the survival of septic rats,decreased histological damage of the lungs,reduced inflammatory cytokines in serum and lung tissues of septic rats and did not increase counts of peritoneal bacteria.In vitro,GW501516 and over-expression of PPARβ/δ attenuated gene expression of TNF-α,IL-1β and IL-6 in alveolar macrophages.Both in vivo and in vitro,PPARβ/δ inhibited the phosphorylation of STAT3.Conclusion PPARβ/δ plays a protective role in sepsis induced ALI via suppressing excessive inflammation.

Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian(Herba Andrographitis Paniculatae) as an anti-inflammatory agent

OBJECTIVE: To systematically evaluate the in vivo antiinflammatory potential of diterpene lactones from Chuanxinlian(Herba Andrographitis Paniculatae)(AP). METHODS: We firstly adopted zebrafish, a novel and ideal animal model for high-throughput drug screening, to investigate the in vivo anti-inflammatory activities of 17 diterpene lactones isolated from AP.RESULTS: The results showed that most of diterpene lactones displayed significant anti-inflammatory effects in lipopolysaccharide microinjection-, copper sulfate exposure-or tail transection-induced zebrafish inflammation models. Moreover, diterpene lactone 3-deoxy-andrographoside(AP-5) was firstly found to attenuate inflammatory responses, which was closely associated with the myeloid differentiation primary response 88/nuclear factor-kappa B and signal transducer and activator of transcription 3 pathways. CONCLUSION: Our research sheds light on the inestimable roles of zebrafish in high-throughput drug screening, elucidates the potent inhibitory effects of diterpene lactones against inflammation and indicates that AP-5 may serve as a potential alternative agent for the treatment of inflammatory diseases.

Tiaobu Feishen therapy inhibits inflammation induced by cigarette smoke extracts in a human monocyte/macrophage cell line

OBJECTIVE: To study the mechanistic effects of Tiaobu Feishen therapy(TBFS) on inflammation induced by cigarette smoke extract(CSE) in a human monocyte/macrophage cell line.METHODS: The human monocyte/macrophage cell line THP-1 was stimulated with 10% CSE in the presence or absence of Bufei Yishen formula(BYF),Bufei Jianpi formula(BJF) and Yiqi Zishen formula(YZF). All formulations contained serum. Pro-inflammatory cytokines were measured in the supernatants using enzyme-linked immunosorbent assay.The activity of STAT3 DNA binding was detected using electrophoretic mobility shift assay and janus kinase/signal transducer and activator of transcription(JAK/STAT) pathway activation was assessed using Western blotting.RESULTS: The results showed that BYF, BJF and YZF treatment strongly decreased the CSE-induced secretion of interleukin(IL)-6, IL-8, tumor necrosis factor-α and matrix metalloproteinase-9 by THP-1 cells. Furthermore, BYF, BJF and YZF treatment attenuated STAT3 DNA binding capacity and JAK2 and STAT3 were shown to be phosphorylated.CONCLUSION: The data revealed that BYF, BJF and YZF effectively inhibited a CSE-induced inflammatory response in THP-1 cells by limiting activation of the JAK2/STAT3 pathway.