Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

STAT3-Dependent Effects of Polymeric Immunoglobulin Receptor in Regulating Interleukin-17 Signaling and Preventing Autoimmune Hepatitis

One-third of patients with autoimmune hepatitis(AIH)have cirrhosis at the time of diagnosis.The relevance of these variables,although unknown,is believed to be critical in AIH because of suspected interactions between the gut microbiome and genetic factors.Dysbiosis of the gut flora and elevated polymeric immunoglobulin receptor(pIgR)levels have been observed in both patients and mouse models.Moreover,there is a direct relationship between pIgR expression and transaminase levels in patients with AIH.In this study,we aimed to explore how pIgR influences the secretion of regenerating islet-derived 3 beta(Reg3b)and the flora composition in AIH using in vivo experiments involving patients with AIH and a concanavalin A-induced mouse model of AIH.Reg3b expression was reduced in pIgR gene(Pigr)-knockout mice compared to that in wild-type mice,leading to increased microbiota disruption.Conversely,exogenous pIgR supplementation increased Reg3b expression and maintained microbiota homeostasis.RNA sequencing revealed the participation of the interleukin(IL)-17 signaling pathway in the regulation of Reg3b through pIgR.Furthermore,the introduction of external pIgR could not restore the imbalance in gut microbiota in AIH,and the decrease in Reg3b expression was not apparent following the inhibition of signal transducer and activator of transcription 3(STAT3).In this study,pIgR facilitated the upregulation of Reg3b via the STAT3 pathway,which plays a crucial role in preserving the balance of the intestinal microbiota in AIH.Through this research,we discovered new molecular targets that can be used for the diagnosis and treatment of AIH.

Immunotherapeutic hydrogel for co-delivery of STAT3 siRNA liposomes and lidocaine hydrochloride for postoperative comprehensive management of NSCLC in a single application

Despite standard treatment for non-small cell lung cancer(NSCLC)being surgical resection,cancer recurrence and complications,such as induction of malignant pleural effusion(MPE)and significant postoperative pain,usually result in treatment failure.In this study,an alginate-based hybrid hydrogel(SOG)is developed that can be injected into the resection surface of the lungs during surgery.Briefly,endoplasmic reticulum-modified liposomes(MSLs)pre-loaded with the signal transducer and activator of transcription 3(STAT3)small interfering RNA and lidocaine hydrochloride are encapsulated in SOG.Once applied,MSLs strongly downregulated STAT3 expression in the tumor microenvironment,resulting in the apoptosis of lung cancer cells and polarization of tumor-associated macrophages towards the M1-like phenotype.Meanwhile,the release of lidocaine hydrochloride(LID)was beneficial for pain relief and natural killer cell activation.Our data demonstrated MSL@LID@SOG not only efficiently inhibited tumor growth but also potently improved the quality of life,including reduced MPE volume and pain relief in orthotopic NSCLC mouse models,even with a single administration.MSL@LID@SOG shows potential for comprehensive clinical management upon tumor resection in NSCLC,and may alter the treatment paradigms for other cancers.

Galectin 2 regulates JAK/STAT3 signaling activity to modulate oral squamous cell carcinoma proliferation and migration in vitro

Background:Galectin 2(LGALS2)is a protein previously reported to serve as a mediator of disease progression in a range of cancers.The function of LGALS2 in oral squamous cell carcinoma(OSCC),however,has yet to be explored,prompting the present study to address this literature gap.Methods:Overall,144 paired malignant tumor tissues and paracancerous OSCC patient samples were harvested and the LGALS2 expression levels were examined through qPCR and western immunoblotting.The LGALS2 coding sequence was introduced into the pcDNA3.0 vector,to enable the overexpression of this gene,while an LGALS2-specific shRNA and corresponding controls were also obtained.The functionality of LGALS2 as a regulator of the ability of OSCC cells to grow and undergo apoptotic death in vitro was assessed through EdU uptake and CCK-8 assays,and flow cytometer,whereas a Transwell system was used to assess migratory activity and invasivity.An agonist of the Janus Kinase 2(JAK2)/Signal Transducer and Activator of Transcription 3(STAT3)pathway was also used to assess the role of this pathway in the context of LGALS2 signaling.Results:Here,we found that lower LGALS2 protein and mRNA expression were evident in OSCC tumor tissue samples,and these expression levels were associated with clinicopathological characteristics and patient survival outcomes.Silencing LGALS2 enhanced proliferation in OSCC cells while rendering these cells better able to resist apoptosis.The opposite was instead observed after LGALS2 was overexpressed.Mechanistically,the ability of LGALS2 to suppress the progression of OSCC was related to its ability to activate the JAK/STAT3 signaling axis.Conclusion:Those results suggest a role for LGALS2 as a suppressor of OSCC progression through its ability to modulate JAK/STAT3 signaling,supporting the potential utility of LGALS2 as a target for efforts aimed at treating OSCC patients.

Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis. Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals. After 4 weeks, OVA atomization and gavage were used for stimulation and treatment. Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74, 3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline. HE, PAS and Masson staining were used to observe the lung histopathological changes in rats. The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA. The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue. Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious. The expressions of IL-6, IL- 23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01);Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased (P< 0.01). The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01). Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

Regulation and function of signal transducer and activator of transcription 3

Signal transducer and activator of transcription 3(STAT3), a member of the STAT family, is a key regulator of many physiological and pathological processes. Significant progress has been made in understanding the transcriptional control, posttranslational modification, cellular localization and functional regulation of STAT3. STAT3 can translocate into the nucleus and bind to specific promoter sequences, thereby exerting transcriptional regulation. Recent studies have shown that STAT3 can also translocate into mitochondria, participating in aerobic respiration and apoptosis. In addition, STAT3 plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. Conditional knockout mouse models make it possible to study the physiological function of STAT3 in specific tissues and organs. This review summarizes the latest advances in the understanding of the expression, regulation and function of STAT3 in physiological and tumorigenic processes.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Downregulation of signal transducer and activator of transcription 3 by sorafenib:A novel mechanism for hepatocellular carcinoma therapy

Hepatocellular carcinoma is one of the most common cancers worldwide,and a leading cause of cancer-related death.Owing to unsatisfactory clinical outcomes under the current standard of care,there is a need to search for and identify novel and potent therapeutic targets to improve patient outcomes.Sorafenib is the first and only approved targeted therapy for the treatment of hepatocellular carcinoma.Besides functioning as a multiple tyrosine kinase,sorafenib also acts via a kinase-independent mechanism to target signal transducer and activator of transcription 3(STAT3) signaling in hepatocellular carcinoma cells.STAT3 is a key regulator of inflammation,cell survival,and tumorigenesis of liver cells,and the high percentage of hepatocellular carcinoma cells with constitutively active STAT3 justifies targeting it for the development of novel therapeutics.Sorafenib inactivates STAT3 and STAT3-related signaling by inducing a conformational change in and releasing the autoinhibition of Src homology region 2 domaincontaining phosphatase-1.This phosphatase negatively regulates STAT3 activity,which leads to the subsequent apoptosis of cancer cells.The novel anti-cancer property of sorafenib will be discussed in this review,not only adding information regarding its mechanism of action but also providing an innovative approach for the development of cancer therapeutics in the future.

Paeoniflorin inhibits human gastric carcinoma cell proliferation through up-regulation of microRNA-124 and suppression of PI3K/Akt and STAT3 signaling

AIM: To examine the potential anti-tumor activity of paeoniflorin in the human gastric carcinoma cell line MGC-803.METHODS: Cell viability and cytotoxic effects in MGC-803 cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay, respectively. Cell apoptosis of MGC-803 cells was measured using flow cytometry,DAPI staining assay and caspase-3 activity assay.Quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the expression of microRNA-124(miR-124) in response to paeoniflorin.The expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt), phospho-Akt(p-Akt) and phospho-signal transducer and activator of transcription3(p-STAT3) were also measured by quantitative RTPCR and Western blot analysis in normal, miR-124 and anti-miR-124 over-expressing MGC-803 cells, treated with paeoniflorin.RESULTS: Paeoniflorin was found to inhibit MGC-803 cell viability in a dose-dependent manner. Paeoniflorin treatment was associated with the induction of apoptosis and caspase-3 activity in MGC-803 cells. Paeoniflorin treatment significantly increased miR-124 levels and inhibited the expression of PI3 K, Akt, p-Akt and p-STAT3 in MGC-803 cells. Interestingly, the over-expression of miR-124 inhibits PI3K/Akt and phospho-STAT3 expressions in MGC-803 cells. PI3 K agonist(IGF-1, 1μg/10 μL) or over-expression of STAT3 reversed the effect of paeoniflorin on the proliferation of MGC-803 cells. Over-expression of anti-miR-124 in MGC-803 cells reversed paeoniflorin-induced up-regulation.CONCLUSION: In summary, the in vitro data suggest that paeoniflorin is a potential novel therapeutic agent against gastric carcinoma, which inhibits cell viability and induces apoptosis through the up-regulation of miR-124 and suppression of PI3K/Akt and STAT3 signaling.

Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

Activated nucleotide binding to the oligonucleotide receptor protein 3（NLRP3） inflammasome is possibly involved in the pathogenesis of Alzheimer＇s disease through oxidative stress and neurogenic inflammation. Low expression of the signal transducer and activator of transcription 3（STAT3） gene may promote the occurrence of neurodegenerative diseases to some extent. To clarify the roles of the NLRP3 inflammasome and STAT3 expression in oxidative stress,（1） SHSY5 Y cells were incubated with 1 mM H2 O2 to induce oxidative stress injury, and the expression of human-cell-specific signal transduction, STAT3-shRNA silencing signal transduction and STAT3 were detected. Cells were pretreated with Ca2＋ chelator BAPATA-AM（0.1 mM） for 30 minutes as a control.（2） Western blot assay was used to analyze the expression of caspase-1, NLRP3, signal transduction and STAT3. Enzyme-linked immunosorbent assay was used to analyze interleukin-1β levels. Flow cytometry was carried out to calculate the number of apoptotic cells. We found that H2 O2 treatment activated NLRP3 inflammasomes and decreased phosphorylation of signal transduction and STAT3 serine 727. BAPTA-AM pretreatment abolished the H2 O2-induced activation of NLRP3 inflammasomes, caspase-1 expression, interleukin-1β expression and apoptosis in SHSY5 Y cells, and had no effect in cells with downregulated STAT3 expression by RNAi. The findings suggest that downregulation of signal transduction and STAT3 expression may enhance the oxidative stress mediated by NLRP3, which may not depend on the Ca2^＋ signaling pathway.

Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion

Signal transducer and activator of transcription（STAT）is a unique protein family that binds to DNA,coupled with tyrosine phosphorylation signaling pathways,acting as a transcriptional regulator to mediate a variety of biological effects.Cerebral ischemia and reperfusion can activate STATs signaling pathway,but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging（DWI）in rats after cerebral ischemia/reperfusion.Here,we established a rat model of focal cerebral ischemia injury using the modified Longa method.DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient.STAT3 protein expression showed no significant change after reperfusion,but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours.Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area.These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

Caprylic Acid Improves Lipid Metabolism, Suppresses the Inflammatory Response and Activates the ABCA1/p-JAK2/pSTAT3 Signaling Pathway in C57BL/6J Mice and RAW264.7 Cells

Objective This study aimed to investigate the effects of caprylic acid(C8:0)on lipid metabolism and inflammation,and examine the mechanisms underlying these effects in mice and cells.Methods Fifty-six 6-week-old male C57BL/6J mice were randomly allocated to four groups fed a highfat diet(HFD)without or with 2%C8:0,palmitic acid(C16:0)or eicosapentaenoic acid(EPA).RAW246.7 cells were randomly divided into five groups:normal,lipopolysaccharide(LPS),LPS+C8:0,LPS+EPA and LPS+cAMP.The serum lipid profiles,inflammatory biomolecules,and ABCA1 and JAK2/STAT3 mRNA and protein expression were measured.Results C8:0 decreased TC and LDL-C,and increased the HDL-C/LDL-C ratio after injection of LPS.Without LPS,it decreased TC in mice(P<0.05).Moreover,C8:0 decreased the inflammatory response after LPS treatment in both mice and cells(P<0.05).Mechanistic investigations in C57BL/6J mouse aortas after injection of LPS indicated that C8:0 resulted in higher ABCA1 and JAK2/STAT3 expression than that with HFD,C16:0 and EPA,and resulted in lower TNF-α,NF-κB mRNA expression than that with HFD(P<0.05).In RAW 264.7 cells,C8:0 resulted in lower expression of pNF-κBP65 than that in the LPS group,and higher protein expression of ABCA1,p-JAK2 and p-STAT3 than that in the LPS and LPS+cAMP groups(P<0.05).Conclusion Our studies demonstrated that C8:0 may play an important role in lipid metabolism and the inflammatory response,and the mechanism may be associated with ABCA1 and the p-JAK2/p-STAT3 signaling pathway.

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Regulation Mechanism of TFP on TGF-β1/STAT3 Signaling Pathway in Immune-mediated Liver Injury in Mice

[Objectives]To study the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP)on immune-mediated liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide(BCG+LPS)in mice,and to explore its action mechanism.[Methods]60 Kunming mice were divided into normal group,model group,control group(bifendate)and TFP low,medium and high dose groups according to random number table method,with 10 mice in each group.On the first day of modeling,mice were injected with 0.2 mL of BCG solution(12.5 mg/mL)through the tail vein,and on the eleventh day,0.2 mL of LPS(37.5μg/mL)were injected into the tail vein to prepare a mouse model of immune-mediated liver injury;from the first day of modeling,the normal group and the model group were administered intragastrically with the corresponding volume of distilled water,and the bifendate group and the TFP high,medium,and low dose groups were administered intragastrically with the corresponding doses once a day for 11 d.After the last time administration,fasting but giving water for 16 h,took blood from eyes,then collected the liver tissue.The levels of alanine transaminase(ALT)and aspartate transaminase(AST)in serum were detected by biochemical method;transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)expression levels in liver tissue were detected by enzyme-linked immunosorbent assay(ELISA);phosphorylated protein tyrosine kinase JAK-2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)protein expression levels were detected by Western Blot method;the degree of liver tissue lesions was detected by HE staining.[Results]Compared with the model group,the levels of ALT and AST in the serum of mice in each dose group of TFP(high dose 600 mg/kg,medium dose 400 mg/kg,and low dose 200 mg/kg)were reduced,and the activities of T-SOD and GSH-Px were increased;the content or expression ofβ1,ICAM-1,IL-6,IL-1βdecreased,and the expression of p-JAK2 and p-STAT3 protein decreased;pathological sections showed that the degree of inflammatory necrosis and the degree of lesions in the liver tissues of each dose group of TFP were reduced by varying degrees.[Conclusions]TFP has a protective effect on BCG+LPS-induced immune-mediated liver injury in mice.The mechanism may be related to regulating the phosphorylation level of JAK2 and inhibiting the inflammatory reaction,thereby regulating the TGF-β1/STAT3 signaling pathway and improving the immune-mediated liver injury.

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.

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction

BACKGROUND Leukemia stem cells(LSCs)are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia(AML),as they are protected by the bone marrow microenvironment(BMM)against conventional therapies.Gossypol acetic acid(GAA),which is extracted from the seeds of cotton plants,exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2.AIM To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism.METHODS In this study,LSCs were magnetically sorted from AML cell lines and the CD34+CD38-population was obtained.The expression of leucine-rich pentatricopeptide repeat-containing protein(LRPPRC)and forkhead box M1(FOXM1)was evaluated in LSCs,and the effects of GAA on malignancies and mitochondrial RESULTS LRPPRC was found to be upregulated,and GAA inhibited cell proliferation by degrading LRPPRC.GAA induced LRPPRC degradation and inhibited the activation of interleukin 6(IL-6)/janus kinase(JAK)1/signal transducer and activator of transcription(STAT)3 signaling,enhancing chemosensitivity in LSCs against conventional chemotherapies,including L-Asparaginase,Dexamethasone,and cytarabine.GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC.Furthermore,GAA induced reactive oxygen species accumulation,disturbed mitochondrial homeostasis,and caused mitochondrial dysfunction.By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC,GAA resulted in the elimination of LSCs.Meanwhile,GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage.CONCLUSION Taken together,the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Leptin Activates STAT3 and ERK1/2 Pathways and Induces Endometrial Cancer Cell Proliferation

Obesity is an established risk factor for endometrial cancer.Leptin,a secreted protein of the ob gene by white adipose tissue,plays an important role in the regulation of food intake and energy consumption in the brain and acts as a potential growth stimulator in normal and neoplastic cancer cells.However,a direct role for leptin in endometrial cancer has not been demonstrated.In the present study,the effect of leptin on the proliferation of Ishikawa endometrial cancer cells was investigated as well as the possible mechanism（s） underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors.The expression of leptin receptor（ObRb） in Ishikawa cells was detected by RT-PCR and Western blotting.The cells after serum starvation,were treated by leptin with various concentrations（0,10,50,100,150 ng/mL） for different durations（6,12,24 h）.The effect of leptin treatment on cell proliferation was examined by MTT assay.Meanwhile,inhibitory effect of Janus tyrosine kinase 2（JAK2）/signal transducer and activator of transcription 3（STAT3） inhibitor AG490 or extracellular signal-regulated kinase 1/2（ERK1/2） inhibitor PD98059 on the proliferation of Ishikawa cells induced by leptin was also studied.Ishikawa cells were treated with 100 ng/mL leptin for various periods（0,20,40,60 min）,and the levels of STAT3 phosphorylation and ERK1/2 phosphorylation were examined by Western blotting.The results showed that leptin induced the phosphorylation of STAT3 and the activation of ERK1/2 in a time-and dose-dependent manner in the Ishikawa endometrial cancer cells.Blocking STAT3 phosphorylation with the inhibitor AG490,or blocking ERK1/2 activation by the specific ERK1/2 kinase inhibitor,PD98059,abolished leptin-induced proliferation of Ishikawa cells.In addition,leptin was found to potently induce the invasion of endometrial cancer cells in a Matrigel invasion assay.Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of STAT3（AG490） and ERK1/2 kinase（PD98059）.These results suggested that leptin promotes endometrial cancer growth and invasiveness by activating STAT3 and ERK1/2 signaling pathways and therefore blocking its action at the receptor level can be a rational therapeutic strategy.

Sorafenib inhibits growth and metastasis of hepatocellular carcinoma by blocking STAT3

AIM: To investigate the inhibitory role and the underlying mechanisms of sorafenib on signal transducer and activator of transcription 3 (STAT3) activity in hepatocellular carcinoma (HCC).METHODS: Human and rat HCC cell lines were treated with sorafenib. Proliferation and STAT3 dephosphorylation were assessed. Potential molecular mechanisms of STAT3 pathway inhibition by sorafenib were evaluated. In vivo antitumor action and STAT3 inhibition were investigated in an immunocompetent orthotopic rat HCC model.RESULTS: Sorafenib decreased STAT3 phosphorylationat the tyrosine and serine residues (Y705 and S727), but did not affect Janus kinase 2 (JAK2) and phosphatase shatterproof 2 (SHP2), which is associated with growth inhibition in HCC cells. Dephosphorylation of S727 was associated with attenuated extracellular signal-regulated kinase (ERK) phosphorylation, similar to the effects of a mitogen-activated protein kinase (MEK) inhibitor U0126, suggesting that sorafenib induced S727 dephosphorylation by inhibiting MEK/ERK signaling. Meanwhile, sorafenib could also inhibit Akt phosphorylation, and both the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and Akt knockdown resulted in Y705 dephosphorylation, indicating that Y705 dephosphorylation by sorafenib was mediated by inhibiting the PI3K/Akt pathway. Finally, in the rat HCC model, sorafenib signifi cantly inhibited STAT3 activity, reducing tumor growth and metastasis.CONCLUSION: Sorafenib inhibits growth and metastasis of HCC in part by blocking the MEK/ERK/STAT3 and PI3K/Akt/STAT3 signaling pathways, but independent of JAK2 and SHP2 activation.

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.

Effects on the STAT3-shRNA in Non-Small-Cell Lung Cancer Therapy: Design, Induction of Apoptosis, and Conjugation with Chitosan-Based Gene Vectors

STAT3 plays a particularly important role in several cancer-related signal transduction pathways.Silencing STAT3 via RNA interference or small molecule inhibitors induces the apoptosis of tumor cells,thereby inhibiting the growth of the tumors.In this study,short-hairpin RNA sequences targeting the STAT3 genes were designed,synthesized,and then connected to pGPU6/GFP/Neo plasmids as the shRNA-expression vectors.The expression of STAT3-shRNA was analyzed by real-time PCR,western blotting,and cell apoptosis assay to study the growth and apoptosis of the cells.Then,the effect of STAT3 knockdown on the NCI-H1650 cells was studied in a tumor mouse model.The results revealed that,after an in vitro transfection,the proliferation of NCI-H1650 cells was inhibited,and the cells were induced to apoptosis.The mRNA and protein expression levels of STAT3 were downregulated in the STAT3-shRNA group.In vivo,the tumor mass and volume in the STAT3-shRNA group were significantly lower than in the other two groups.Both the in vivo and in vitro results demonstrated a long-period inhibiting effect on NSCLC,especially in vivo,when the tumor inhibition rate could reach 50%in the STAT3-shRNA group,which is an exciting outcome.Moreover,the study of the conjugation of STAT3-shRNA and chitosan-based vectors revealed that they could be combined steadily with good cytocompati-bility and transfection efficiency.These results together provide convincing evidence for the application of STAT3-shRNA used in the treatment of non-small lung cancer,which could be a promoting prospect for the development of gene therapy.