Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

Clinical Manifestation of Calreticulin Gene Mutations in Essential Thrombocythemia without Janus Kinase 2 and MPL Mutations： A Chinese Cohort Clinical Study

Background： Recently, calreticulin （CALR） gene mutations have been identified in patients with essential thrombocythemia （ET）. A high-frequency of ET cases without Janus kinase 2 （JAK2） mutations contain CALR mutations and exhibit clinical characteristics different from those with mutant JAK2. Thus, we investigated the frequency and clinical features of Chinese patients of Han ethnicity with CALR mutations in ET. Methods： We recruited 310 Chinese patients of Han ethnicity with ET to analyze states of CALR, JAK2 V617F, and MPLW5 15 mutations by polymerase chain reaction and direct sequencing. We analyzed the relationship between the mutations and clinical features. Results： CALR, JAK2V617E and MPLW515 mutations were detected in 30% （n = 92）, 48% （n = 149）, and 1% （n = 4） of patients with ET, respectively. The mutation types of CALR involved deletion and insertion of base pairs. Most of them were Type 1 （52-bp deletion） and Type 2 （5-bp insertion, TTGTC） mutations, leading to de1367fs46 and ins385fs47, respectively. The three mutations were exclusive. Clinically, patients with mutated CALR had a lower hemoglobin level, lower white blood cell （WBC） count, and higher platelet count compared to those with mutated JAK2 （P 〈 0.05）. Furthermore, a significant difference was found in WBCs between wild-type patients （triple negative for JAK2, MPL, and CALR mutations） and patients with JAK2 mutations. Patients with CA LR mutations predominantly clustered into low or intermediate groups according to the International Prognostic Score of thrombosis for ET （P 〈 0.05）. Conclusions： CALR mutations were frequent in Chinese patients with ET, especially in those without JAK2 or MPL mutations. Compared withJAK2 mutant ET, CALR mutant ET showed a different clinical manifestation and an unfavorable prognosis. Thus, C4LR is a potentially valuable diagnostic marker and therapeutic target in ET.

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.

IgG4-related sclerosing cholangitis associated with essential thrombocythemia:A case report

BACKGROUND The complexity of immunoglobulin G4(IgG4)-related diseases and their potential connection to hematologic malignancies remains unclear.This article provided a review of the diagnosis and treatment of a patient with IgG4-related sclerosing cholangitis(SC)and essential thrombocythemia(ET),along with an analysis of relevant literature to enhance comprehension of this disease.CASE SUMMARY A 56-year-old male was admitted to two hospitals with deteriorating jaundice and pruritus prior to hospitalization.Beyond our expectations,the patient was first diagnosed with IgG4-SC and ET with the Janus kinase 2 V617F mutation.Interestingly,the administration of acetate prednisone significantly resulted in improvements in both IgG4-SC and ET.Clinicians need to pay attention to immune disorders and inflammation as they contribute to the development of various disease phenotypes.CONCLUSION When IgG4-SC is suspected without histopathological evidence,diagnostic therapy and long-term regular follow-up can lead to positive treatment outcomes.Clinicians should be mindful of the potential presence of concurrent hematologic diseases in patients with immune disorders.

Hepatocellular carcinoma-derived exosomal miRNA-761 regulates the tumor microenvironment by targeting the SOCS2/JAK2/STAT3 pathway

BACKGROUND:Exosomes and exosomal microRNAs have been implicated in tumor occurrence and metastasis.Our previous study showed that microRNA-761(miR-761)is overexpressed in hepatocellular carcinoma(HCC)tissues and that its inhibition affects mitochondrial function and inhibits HCC metastasis.The mechanism by which exosomal miR-761 modulates the tumor microenvironment has not been elucidated.METHODS:Exosomal miR-761 was detected in six cell lines.Cell counting kit-8(CCK-8)and transwell migration assays were performed to determine the function of exosomal miR-761 in HCC cells.The luciferase reporter assay was used to analyze miR-761 target genes in normal fi broblasts(NFs).The inhibitors AZD1480 and C188-9 were employed to determine the role of the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in the transformation of cancer-associated fi broblasts(CAFs).RESULTS:In this study,we characterized the mechanism by which miR-761 reprogrammed the tumor microenvironment.We found that HCC-derived exosomal miR-761 was taken up by NFs.Moreover,HCC exosomes aff ected the tumor microenvironment by activating NFs via suppressor of cytokine signaling 2(SOCS2)and the JAK2/STAT3 signaling pathway.CONCLUSIONS:These results demonstrated that exosomal miR-761 modulated the tumor microenvironment via SOCS2/JAK2/STAT3 pathway-dependent activation of CAFs.Our fi ndings may inspire new strategies for HCC prevention and therapy.

Fructose 1,6-diphosphate alleviates myocardial ischemia reperfusion injury in rats through JAK2/STAT3 pathway

Objective: To study the effect of fructose 1,6-diphosphate(FDP) on myocardial ischemia reperfusion injury in rats and its molecular mechanism.Methods: Male SPF SD rats were selected as experimental animals and randomly divided into four groups.Sham group received sham operation, I/R group were made into myocardial ischemia reperfusion injury models, FDP group were made into myocardial ischemia reperfusion injury models and then were given FDP intervention, and FDP+AG490 group were made into myocardial ischemia reperfusion injury models and then were given FDP and JAK2 inhibitor AG490 intervention.Results: CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of I/R group were significantly higher than those of Sham group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissues were significantly lower than those of Sham group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP group were significantly lower than those of I/R group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissue were significantly higher than those of I/R group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP+AG490 group were significantly higher than those of FDP group whereas Bcl-2 protein expression in myocardial tissue was significantly lower than that of FDP group.Conclusion: FDP could reduce the myocardial ischemia reperfusion injury in rats by activating the JAK2/STAT3 pathway.

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.

Electroacupuncture effect on neurological behavior and tyrosine kinase-JAK2 in rats with focal cerebral ischemia

OBJECTIVE:Electroacupuncture effect on neurological behavior and the expression of tyrosine kinase Janus kinase 2(JAK 2) of ischemic cortex in rats with the focal cerebral ischemia were investigated in this study.METHODS:The model of focal cerebral ischemia was established by the heat-coagulation induced the occlusion of the middle cerebral artery.The electro-acupuncture was applied on Baihui(GV 20) and Dazhui(GV 14),and AG490 was applied by intracerebroventricular infusion.The expressions of JAK2 mRNA and phospharylatedJAK2(p-JAK2) in the ischemic cortex were observed by in situ hybridization and western blotting.RESULTS:The expressions of JAK2 mRNA and p-JAK2 were rarely found in sham surgery group.In model group,the expression of JAK2 mRNA and JAK2 phosphorylation had increased.After 1 day of cerebral ischemia,the expression had reached its peak.After cerebral ischemia,the expressions of JAK2 mRNA and p-JAK2 were consistent with the neurological deficit score.Electroacupuncture treatment and AG490 intervention were able to improve the neurological deficit score after cerebral ischemia,and down-regulate the expressions of JAK2 mRNAand JAK2 phosphorylation.CONCLUSION:After cerebral ischemia,the excessive expressions of JAK2 and the JAK2 phosphorylation would be one of mechanisms by which the brain injury got worse.The therapy of electro-acupuncture could reduce the expression of JAK2,and inhibit JAK2 phosphorylated activation,so as to block the abnormal activation of signal transduction pathway which was induced by JAK2.

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.

Treatment of gastric varices with partial splenic embolization in a patient with portal vein thrombosis and a myeloproliferative disorder

Therapeutic options for gastric variceal bleeding in the presence of extensive portal vein thrombosis associated with a myeloproliferative disorder are limited.We report a case of a young woman who presented with gastric variceal bleeding secondary to extensive splanchnic venous thrombosis due to a Janus kinase 2 mutation associated myeloproliferative disorder that was managed effectively with partial splenic embolization.

Profilin-1 is involved in macroangiopathy induced by advanced glycation end products via vascular remodeling and inflammation

BACKGROUND The accumulation of advanced glycation end products(AGEs)have been implicated in the development and progression of diabetic vasculopathy.However,the role of profilin-1 as a multifunctional actin-binding protein in AGEs-induced atherosclerosis(AS)is largely unknown.AIM To explore the potential role of profilin-1 in the pathogenesis of AS induced by AGEs,particularly in relation to the Janus kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3)signaling pathway.METHODS Eighty-nine individuals undergoing coronary angiography were enrolled in the study.Plasma cytokine levels were detected using ELISA kits.Rat aortic vascular smooth muscle cells(RASMCs)were incubated with different compounds for different times.Cell proliferation was determined by performing the MTT assay and EdU staining.An AGEs-induced vascular remodeling model was established in rats and histological and immunohistochemical analyses were performed.The mRNA and protein levels were detected using real-time PCR and Western blot analysis,respectively.In vivo,shRNA transfection was performed to verify the role of profilin-1 in AGEs-induced proatherogenic mediator release and aortic remodeling.Statistical analyses were performed using SPSS 22.0 software.RESULTS Compared with the control group,plasma levels of profilin-1 and receptor for AGEs(RAGE)were significantly increased in patients with coronary artery disease,especially in those complicated with diabetes mellitus(P<0.01).The levels of profilin-1 were positively correlated with the levels of RAGE(P<0.01);additionally,the levels of both molecules were positively associated with the degree of coronary artery stenosis(P<0.01).In vivo,tail vein injections of AGEs induced the release of proatherogenic mediators,such as asymmetric dimethylarginine,intercellular adhesion molecule-1,and the N-terminus of procollagen III peptide,concomitant with apparent aortic morphological changes and significantly upregulated expression of the profilin-1 mRNA and protein in the thoracic aorta(P<0.05 or P<0.01).Downregulation of profilin-1 expression with an shRNA significantly attenuated AGEs-induced proatherogenic mediator release(P<0.05)and aortic remodeling.In vitro,incubation of vascular smooth muscle cells(VSMCs)with AGEs significantly promoted cell proliferation and upregulated the expression of the profilin-1 mRNA and protein(P<0.05).AGEs(200μg/mL,24 h)significantly upregulated the expression of the STAT3 mRNA and protein and JAK2 protein,which was blocked by a JAK2 inhibitor(T3042-1)and/or STAT3 inhibitor(T6308-1)(P<0.05).In addition,pretreatment with T3042-1 or T6308-1 significantly inhibited AGEs-induced RASMC proliferation(P<0.05).CONCLUSION AGEs induce proatherogenic events such as VSMC proliferation,proatherogenic mediator release,and vascular remodeling,changes that can be attenuated by silencing profilin-1 expression.These results suggest a crucial role for profilin-1 in AGEs-induced vasculopathy.

Mediating effect of dopamine D3 receptors on Jak2 and GABAAα1 expression in mouse brains induced by cocaine

Background Cocaine addiction may involve complex neuroadaptations, including many changes of genes expression. Dopamine D3 receptors play an important role in cocaine addiction; however, its role in cocaine induced gene expression change is poorly understood. To identify the changes in gene expression induced by repeated cocaine exposure through D3 dopamine receptors, we compared the expression of four molecules： Janus kinase 2 （Jak2）, g-aminobutanoic acid receptor subunit alpha 1 （GABAAα1）, glutamate receptor AMPA3 alpha 3 （GluR 3） and stromal cell derived factor 1 （SDF1）. These four have been implicated in mediating the actions of cocaine in the nucleus accumbens （NAc） and caudoputamen （CPu） in mice after acute and repeated cocaine exposure. Methods For the acute and repeated injections, the mice were divided into four groups： 30 mg/kg cocaine, nafadotride 0.5 mg/kg ＋ cocaine 30 mg/kg, nafadotride 0.5 mg/kg, and saline as the basal group. The expression of Jak2, GABAAα1, GluR 3 and SDF1 were assayed by Western blot, quantitative real-time RT-PCR and immunohistochemistry. Results Twenty-four hours after seven consecutive days of repeated cocaine exposure, the expression of GABAAα1 decreased in cocaine group compared with basal line and further decreased in the cocaine ＋ nafadotride group and remained at basal level in the nafadotride group. Similarly, the Jak2 expression decreased in cocaine group compared with base line. However, the levels of Jak2 increased in cocaine ＋ nafadotride group compared with cocaine group, while remained at basal level in nafadotride group. Conclusions GABAAα1 and Jak2 may be involved in chronic cocaine induced neuroadaptations. D3 dopamine receptors play an important role in the expression of these genes.

Effect of electroacupuncture on JAK2/STAT3 pathway in synovial tissues of rats with rheumatoid arthritis

Objective: To observe the effect of electroacupuncture (EA) on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in knee joint synovial tissues of rats with rheumatoid arthritis (RA) and to explore the action mechanism of EA on RA. Methods: Twelve of the 48 SPF male Sprague-Dawley (SD) rats were assigned to a normal group by the random number table method. The remaining 36 rats were subjected to RA model preparation by intradermal injection of the Freund's complete adjuvant into the right hind foot pad of each rat under sterile conditions. After the model was successfully prepared, rats were then divided into a model group, a drug group and an EA group according to a random number table method (n=12). Rats in the drug group were treated with 2 mL aqueous solution of tripterygium glycosides [8.1 mg/(kg?bw)];rats in the EA group were treated with EA at bilateral Yanglingquan (GB 34) and Zusanli (ST 36), for 30 min each time;rats in the normal group and the model group were placed in a special rat fixation tank for 30 min each time, and received the same dose of normal saline as those in the drug group. Rats in all groups received intervention once a day for 4 weeks. Diameter of rat ankle joint and rat arthritis index were measured before and after the intervention. At the end of the experiment, the expressions of phospho-JAK2 and phospho-STAT3 were determined by immunohistochemistry. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect JAK2 and STAT3 mRNAs expressions. Results: After the model was produced, the arthritis index >2 was considered successful in model preparation. Compared with the model group, the ankle joint diameters and arthritis indexes of rats in the drug group and the EA group were significantly lower (all P<0.01);immunohistochemical staining cells with phospho-JAK2 and phospho-STAT3 were significantly decreased (all P<0.01);the expression levels of JAK2 and STAT3 mRNAs were decreased with statistical differences (all P<0.01). There were no significant differences between the EA group and the drug group (all P>0.05). Conclusion: EA can alleviate the inflammatory response of RA rats, improve their pathological conditions, reduce the expressions of phospho-JAK2 and phospho-STAT3 in the synovial tissue of knee joint, and decrease the expressions of JAK2 and STAT3 mRNAs. The therapeutic effect of EA is comparable to that of the tripterygium glycosides. The mechanism of EA treatment may be related to the inactivation of the JAK2/STAT3 pathway.

Effects of Danshen Injection(丹参注射液) on Inhibiting Proliferation and Inducing Apoptosis through Down-Regulation of Mutant JAK2 Gene and Its Protein Phosphorylation in Human Erythroid Leukemic Cells

Objective： To explore the effects of Danshen Injection （丹参注射液） on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic （HEL） cells. Methods： The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （M＇l-r） method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide （PI） staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez （JAK2） gene and phosphorylation-JAK2 （P-JAK2） protein were detected by allele specific-polymerase chain reaction and Western blot. ]Results： The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46 ± 2.31% to 50.20 ± 5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection. Conclusion： Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.

Regulatory Effect of JAK2/STAT3 on the Immune Function of Endotoxin-tolerant Dendritic Cells and its Involvement in Acute Liver Failure

Background and Aims:Acute liver failure(ALF)is a potentially fatal clinical syndrome with no effective treatment.This study aimed to explore the role of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)pathway in modulating the phenotype and immune function of endotoxin-tolerant dendritic cells(ETDCs).In addition,we explored the use of EDTCs in an experimental model of ALF and investigated the associated mechanisms.Methods:In the in vitro experiment,ETDCs were transfected with adenovirus to induce SOCS1^(+/+)ETDCs and SOCS1^(−/−)ETDCs.Thereafter,costimulatory molecules and mixed lymphocyte reaction were assessed.Experimental mice were randomly divided into normal control,ALF,ALF+mock-ETDCs,ALF+SOCS1^(+/+)ETDCs,ALF+AG490,and ALF+AG490+SOCS1^(+/+)ETDCs groups.We examined the therapeutic effect of adoptive cellular immunotherapy by tail-vein injection of target ETDCs 12 h before ALF modeling.AG490,a JAK2/STAT3 inhibitor,was used in the in vivo experiment to further explore the protective mechanism of SOCS1^(+/+)ETDCs.Results:Compared with control ETDCs,SOCS1^(+/+)ETDCs had lower expression of costimulatory molecules,weaker allostimulatory ability,lower levels of IL-6 and TNF-αexpression and higher IL-10 secretion.SOCS1^(−/−)ETDCs showed the opposite results.In the in vivo experiments,the ALF+SOCS1^(+/+)ETDCs and ALF+AG490+SOCS1^(+/+)ETDCs groups showed less pathological damage and suppressed activation of JAK2/STAT3 pathway.The changes were more pronounced in the ALF+AG490+SOCS1^(+/+)ETDCs group.Infusion of SOCS1^(+/+)ETDCs had a protective effect against ALF possibly via inhibition of JAK2 and STAT3 phosphorylation.Conclusions:The SOCS1 gene had an important role in induction of endotoxin tolerance.SOCS1^(+/+)ETDCs alleviated lipopolysaccharide/D-galactosamine-induced ALF by downregulating the JAK2/STAT3 signaling pathway.

The m6A reader YTHDF2 alleviates the inflammatory response by inhibiting IL-6R/JAK2/STAT1 pathway-mediated high-mobility group box-1 release

Background:Sepsis is a common severe complication in major burn victims and is characterized by a dysregulated systemic response to inflammation.YTH domain family 2(YTHDF2),a wellstudied N6-methyladenosine(m6A)reader that specifically recognizes and binds to m6A-modified transcripts to mediate their degradation,is connected to pathogenic and physiological processes in eukaryotes,but its effect on sepsis is still unknown.We aimed to discover the effects and mechanisms of YTHDF2 in sepsis.Methods:Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and western blot analyses were used to measure the expression of YTHDF2,the interleukin 6 receptor(IL-6R),high-mobility group box-1(HMGB1),Janus kinase 2(JAK2)and signal transducer and activator of transcription 1(STAT1)under different in vitro conditions.Enzyme-linked immunosorbent assays were utilized to evaluate the expression of HMGB1,IL-6,IL-1βand tumor necrosis factor-α.To confirm that YTHDF2 specifically targets IL-6R mRNA,RNA immunoprecipitation and dual-luciferase reporter assays were performed.Finally,we utilized a mouse model of lipopolysaccharide(LPS)-induced sepsis to verify the effects of YTHDF2 in vivo.Results:According to our findings,YTHDF2 was expressed at a low level in peripheral blood mononuclear cells from septic mice and patients as well as in LPS-induced RAW264.7 cells.Overexpression of YTHDF2 alleviated the inflammatory response by inhibiting HMGB1 release and JAK2/STAT1 signalling in LPS-stimulated cells.Mechanistically,YTHDF2 suppressed JAK2/STAT1 signalling by directly recognizing the m6A-modified site in IL-6R and decreasing the stability of IL-6R mRNA,thereby inhibiting HMGB1 release.In vivo experiments showed that YTHDF2 played a protective role in septic mice by suppressing the IL-6R/JAK2/STAT1/HMGB1 axis.Conclusions:In summary,these findings demonstrate that YTHDF2 plays an essential role as an inhibitor of inflammation to reduce the release of HMGB1 by inhibiting the IL-6R/JAK2/STAT1 pathway,indicating that YTHDF2 is a novel target for therapeutic interventions in sepsis.

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.

A Study on the Mechanism of the Protective Effect of GuangeFang on Sepsis-Associated Acute Kidney Injury

Objective:The objective of this study was to explore the mechanism of Guan Gefang(GGF);raw rhubarb 30 g,cassia arboreal 30 g,raw oyster 30 g,ground elm 60 g,and dandelion 30 g.kidney protection.Materials and Methods:Thirty-six Sprague Dawley rats were randomly divided into a control group(Group N),a sepsis control group(Group S),and a sepsis+GGF group(Group G).For Group N,8 ml/kg 0.9%NaCl was used as an enema;for Group S,cecal ligation and puncture(CLP)method was used for modeling and 8 ml/kg 0.9%NaCl was used as an enema;and Group G,CLP was used for modeling and 8 ml/kg GGF was used as an enema.All of the enemas were applied once daily for 4 days.The indices of serum creatinine(SCr),blood urea nitrogen(BUN),uric acid(UA),mammalian target of rapamycin(mTOR),Janus kinase 2(JAK2)were compared across each group.Results:Compared to Group S,Group G had lower levels of SCr,BUN,and UA(P<0.05),while the activities of mTOR and JAK2 were significantly inhibited.Conclusion:GGF may have inhibited the JAK2 or mTOR signaling pathways to protect the rats’kidneys,which had sepsis-associated acute kidney injury.