Shujin Jiannao Prescription alleviates inflammation in the cerebral cortex of rats with hypoxic-ischemic cerebral palsy / 中国组织工程研究

BACKGROUND:Shujin Jiannao Prescription is an empirical formula for the treatment of cerebral palsy in Dongzhimen Hospital,Beijing University of Chinese Medicine,with clear clinical efficacy,but the specific mechanism needs to be elucidated. OBJECTIVE:To explore the possible mechanism of Shujin Jiannao Prescription in treating cerebral palsy. METHODS:Sixty-four 7-day-old Sprague-Dawley rats were randomly divided into a normal group(n=12)and a model group(n=52).An animal model was established by the Rice-Vannucci method.After successful modeling,52 model rats were randomly divided into control model group(n=12),minocycline group,and the low-,medium-,and high-dose groups of the Shujin Jiannao Prescription(n=10 per group).Rats in the minocycline group were given 40 mg/kg·d minocycline by gavage;rats in the low-,medium,and high-dose groups were given 4,8,and 16 g/kg·d Shujin Jiannao Prescription granules by gavage,respectively;and rats in the normal group and control model group were given an equal dose of normal saline by gavage.Medication in each group was given once a day for 1 week.The rats in each group were evaluated behaviorally using suspension test,abnormal involuntary movement score,and Bederson score.The pathological changes of the cerebral cortex were observed by hematoxylin-eosin staining.The levels of tumor necrosis factor α,interleukin 1β,and interleukin 10 in the cerebral cortex were determined using ELISA.The positive expressions of Janus kinase 2(JAK2),phosphorylated Janus kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)in the cerebral cortex were detected using immunohistochemistry.The protein expression levels of JAK2,p-JAK2,and p-STAT3 were detected using western blot. RESULTS AND CONCLUSION:Compared with the normal group,the suspension test score and involuntary movement score were decreased in the control model group(P＜0.01 or P＜0.05).The pathological results showed structural disruption of nerve cells,formation of large numbers of vacuoles,cell swelling,and increased intercellular space in the control model group.In addition,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were significantly increased(P＜0.01),the expression of interleukin 10 was decreased(P＜0.05),and the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly increased(P＜0.01)in the control model group compared with the normal group.Compared with the model group,minocycline and Shujin Jiannao Prescription at each dose could improve the behavioral indexes of rats(P＜0.01 or P＜0.05)and ischemic-hypoxic pathological changes were attenuated,with only a small amount of necrotic nerve cells and a few vacuoles,and reduced intercellular space.Moreover,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were decreased in each drug group compared with the control model group(P＜0.05),while the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly decreased(P＜0.01).The most obvious improvement was observed in the high-dose Shujin Jiannao Prescription group.To conclude,Shujin Jiannao Prescription can inhibit inflammation in the cerebral cortex of rats with hypoxic-ischemic brain injury.The mechanism may be related to the regulation of the JAK2/STAT3 signaling pathway.

Effects of Electroacupuncture on Post Stroke Spasticity Rats Based on IL-6/JAK2/STAT3 Signaling Pathway / 中国中医药信息杂志

Objective To observe the effects of electroacupuncture on central inflammatory response and neurotransmitter release in rats with post stroke spasticity(PSS);To exploring the mechanism in treating PSS based on IL-6/JAK2/STAT3 signaling pathway.Methods Totally 30 male SD rats were randomly divided into sham-operation group,model group and electroacupuncture group,with 10 rats in each group.The PSS model was prepared by the method of suture and N-methyl-D-aspartic acid receptor injection into internal capsule.The rats in the electroacupuncture group were electroacupulated on the affected side of the body at"Quchi"and"Yanglingquan"for 30 min/d for consecutive 7 d.The sham-operation group and the model group were only fixed without any interventions.The Zea Longa neurological function score and the modified Ashworth muscle tension score were evaluated before and after treatment in each group;the pathological changes of the cortex on the ischemic side were observed by HE staining;the contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and γ-aminobutyric acid(GABA)in cortex on the ischemic side were detected by ELISA;the content of glutamate(Glu)was detected by biochemical kit;Western blot was used to detect the expressions of tyrosine kinase 2(JAK2),p-JAK2,signal transduction and transcription activating factor 3(STAT3)and p-STAT3 protein in ischemic cortex;RT-PCR was used to detect the mRNA expressions of JAK2 and STAT3 in ischemic cortex.Results Compared with the sham-operation group,neurological function score and muscle tension score significantly increased in the model group(P<0.01),with disorganized neurons in cerebral cortex,nucleus accumbens,the contents of IL-6,TNF-α and Glu significantly increased,the content of GABA significantly decreased(P<0.01),and p-JAK2,p-STAT3 proteins and JAK2 and STAT3 mRNA expression significantly increased(P<0.01,P<0.05).Compared with the model group,the neurological function score and muscle tension score of rats in the electroacupuncture group were significantly decreased(P<0.05),the degree of neuronal damage in cerebral cortex was reduced,the cell contour was clear,the content of IL-6,TNF-α and Glu were significantly decreased,and the content of GABA significantly increased(P<0.05,P<0.01),p-JAK2,p-STAT3 protein and JAK2,STAT3 mRNA expression significantly decreased(P<0.01).Conclusion Electroacupuncture may alleviate central inflammatory response and improve limb spasticity of PSS model rats by inhibiting the IL-6/JAK2/STAT3 signaling pathway.

Exploring the Mechanism of Anti-asthma Effect of Fujiu Patch on Modulation of Th17/Treg Immune Balance Based on IL-6/STAT3 and IL-2/STAT5 Signaling Pathways / 中药新药与临床药理

Objective This study aims to investigate the regulatory effects of Fujiu Patch(composed of Sinapis Semen,Kansui Radix,Corydalis Rhizoma and Asari Radix et Rhizoma)on the CD4+ T helper 17 cell(Th17)/CD4+CD25+ regulatory T cell(Treg)balance in asthmatic rats via the signal pathway of IL-6/signal transducer and activator of transcription 3(STAT3)as well as IL-2/signal transducer and activator of transcription 5(STAT5),and to reveal its anti-asthma mechanisms.Methods An experimental asthma model was constructed by ovalbumin(OVA)combined with aluminum hydroxide sensitization and challenge,and then the rats were administered with Fujiu Patch at Dazhui(DU14),Feishu(BL13)and Shenshu(BL23)points for 4 hours each time,once every other day for 7 times.Immunohistochemistry was used to detect the positive expressions of Th17 specific cytokine(IL-17)and Treg transcription factor(Foxp3)in rat lung tissue.The percentage of Th17 and Treg cells in peripheral blood was examined by flow cytometry analysis,and the expressions of IL-6/STAT3 and IL-2/STAT5 pathway-related proteins in lung tissue were assayed with Western Blot.Results Compared to the model group,IL-17 positive expression in the rat lung showed a significant reduction in the Fujiu Patch group(P＜0.01),while the positive expression of Foxp3 was obviously increased(P＜0.05).Meanwhile,the protein expression levels of IL-6 and phospho-STAT3 were were significantly declined(P＜0.01),and the protein expression levels of IL-2 and phospho-STAT5 were were significantly elevated(P＜0.01).However,there was no significant alteration in the total protein expressions of STAT3 and STAT5(P＞0.05).Furthermore,the proportion of Th17 cells in peripheral blood of rats in the Fujiu Patch group was lower than that in the model group,while the proportion of Treg cells was higher than that in the model group.Statistically-significant differences were observed(all P＜0.01).Conclusion These findings indicate that Th17/Treg immune imbalance occurs in asthmatic rat.Fujiu Patch may exert anti-asthma effects via inhibiting the expression of IL-6,downregulating the expression of phospho-STAT3,diminishing the level of IL-17-producing Th17 cells,as well as increasing the expressions of IL-2-mediated STAT5 phosphorylation,raising the level of Foxp3-expressing Treg cells,promoting Th17/Treg balance and suppressing immune responses in rat with asthma.

Folic Acid-Modified Liposome Quercetin Induces Apoptosis of Triple-Negative Breast Cancer Cells via Mitochondrial Apoptosis Mediated by JAK2/STAT3 Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect of folic acid–modified liposome quercetin (FLQ) on the proliferation and apoptosis of triple negative breast cancer (TNBC) cells and explore its underlying mechanism. Methods CCK-8 was used to detect the effect of FLQ on TNBC cell viability. Colony formation assay was conducted to detect the effect of FLQ on TNBC cell proliferation. Flow cytometry was performed to detect the effect of FLQ on TNBC cell apoptosis, the levels of intracellular ROS, and mitochondrial membrane potential. Western blot analysis was conducted to detect the expression levels of JAK2/STAT3 signaling pathway-related and apoptosis-related proteins. Results FLQ inhibited the proliferation and promoted the apoptosis of MDA-MB-231 cells (P=0.023, P<0.001). It promoted mitochondrial membrane potential collapse and increased the intracellular ROS levels of MDA-MB-231 cells (P=0.003, P=0.034); inhibited the phosphorylation levels of JAK2 and STAT3; upregulated the expression levels of the proapoptotic proteins Bax, Bak, cytochrome C, and Cleaved-Caspase-3 (P<0.001, P<0.001); and downregulated the expression levels of the antiapoptotic proteins Bcl2 and Bcl-xL (P=0.037, 0.028). Conclusion FLQ inhibits the proliferation and induces the apoptosis of MDA-MB-231 cells. These effects may be related to the activation of the mitochondrial apoptosis pathway through the inhibition of the JAK2/STAT3 signaling pathway.

Baitouweng Tang Inhibits Growth of Esophageal Cancer Cells Through BUB1/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Baitouweng Tang in inhibiting the growth of esophageal cancer （EC） cells by regulating budding uninhibited by benzimidazoles 1 （BUB1）/signal transducer and activator of transcription 3 （STAT3） signaling pathway. MethodGene chip technology was used to explore the differential gene expression between esophageal cancer tissues and normal tissues and identified differentially expressed genes. The differentially expressed genes were analyzed by bioinformatics methods. EC cells were treated with 25， 50， 100， 200， 400， 800 mg·L-1 Baitouweng Tang. EC cell viability was detected by Thiazolyl Blue （MTT） colorimetry. Cell cycle and apoptosis were measured by flow cytometry. The expression of BUB1 was measured by real time quantitative polymerase chain reaction （Real-time PCR）. The protein levels of BUB1， STAT3， phosphorylated （p）-STAT3， Cyclin B1 （CCNB1）， cyclin-dependent kinase 1 （CDK1）， B-cell lymphoma-2 （Bcl-2）， cysteinyl aspartate-specific proteinase（Caspase）-3， and Caspase-9 were measured by Western blot. The migration and invasion abilities of the cells were measured by wound-healing and Transwell invasion assays. ResultDifferentially expressed genes were primarily involved in biological processes， signaling pathways， and network construction related to cell mitosis， with BUB1 identified as a key core gene. Compared with the control group， Baitouweng Tang inhibited BUB1 expression （P<0.05，P<0.01）. In vitro experiments showed that compared with the control group， Baitouweng Tang could significantly inhibit the growth （P<0.05，P<0.01）， migration and invasion （P<0.05，P<0.01） of EC cells， induce apoptosis （P<0.05，P<0.01）， and cause G2/M phase increase （P<0.01）. After treatment with Baitouweng Tang， compared with the results in the control group， the expression of Caspase-3， and Caspase-9 in EC cells increased significantly （P<0.05，P<0.01）， while the expression of Bcl-2， BUB1， CCNB1， and CDK1 decreased significantly （P<0.05，P<0.01）. Moreover， the STAT3 signaling pathway was also found to play an important role in this process. ConclusionBaitouweng Tang may inhibit the growth of EC cells by downregulating BUB1 and mediating the STAT3 signaling pathway.

Improvement of Colonic Mucosa Inflammatory Response in Mice with Ulcerative Colitis by Xiezhuo Jiedu Recipe Through miRNA-155-5p/JAK2/STAT3 Pathway / 中国实验方剂学杂志

ObjectiveThe differential expression of microRNAs （miRNAs） between the active stage and the remission stage of ulcerative colitis （UC） was analyzed by bioinformatics method， and the regulatory relationship was constructed by screening the differentially expressed genes （DEGs）. The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database （GEO）， and the most differentially expressed miRNAs were screened by GEO2R， Excel， and other tools as research objects. TargetScan， miRTarbase， miRDB， STRING， TRRUST， and Matescape databases were used to screen key DEGs， predict downstream transcription factors （TFs）， gene ontology （GO）， and conduct Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor （SOCS1）， phosphorylated transcriptional signal transductor and activator 3 （p-STAT3）， phosphorylated Janus kinase 2 （p-JAK2）， and retinoic acid-associated orphan receptor-γt （ROR-γt）. The expression levels of transforming growth factor-β （TGF-β）， interleukin-17 （IL-17）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） in serum were detected by enzyme linked immunosorbent assay （ELISA）. ResultThe GSE48957 dataset was screened from the GEO database， and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation， as well as signaling pathways such as stem cell signaling pathway， IL-17 signaling pathway， and helper T cell 17 （Th17） cell differentiation. The Matescape database was used to screen out 10 key DEGs， among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3， p-JAK2， and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice， up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10， increase the level of anti-inflammatory factors， and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice， inhibit the phosphorylation of STAT3， inhibit the differentiation of CD4+ T cells into Th17 cells， reduce the levels of pro-inflammatory factors （IL-17 and IL-6）， and increase the levels of anti-inflammatory factors （TGF-β and IL-10）. As a result， the inflammation of colon mucosa in UC mice was alleviated.

Effects of Maxing Shigan Decoction on IL-6/STAT3 Signaling Pathway and TRPV1 Receptor in Rats with Cough Variant Asthma / 广州中医药大学学报

Objective To investigate the therapeutic effects and mechanisms of Maxing Shigan Decoction on cough variant asthma(CVA)rats.Methods Sixty rats were randomly divided into normal group,model group,low and high dose groups of Maxing Shigan Decoction,and high-dose of Maxing Shigan Decoction + signal transducer and activator of transcription 3(STAT3)activator Colivelin(Col)group,12 rats in each group.Except for the normal group,the CVA model was constructed by intraperitoneal injection of ovalbumin combined with moxa fumigation in all other groups of rats.After the corresponding treatment,the rats were observed for signs and cough counts,airway resistance(RE)was detected by pulmonary function meter,eosinophils(EOS)were counted by Diff-Quik staining,histopathological features of the lungs and bronchial tubes were observed by hematoxylin-eosin(HE)staining method,and the lung tissues were detected by enzyme-linked immunosorbent assay(ELISA)for monocyte chemotactic protein 1(MCP-1),and tumor necrosis factor α(TNF-α),and the protein expression levels of interleukin 6(IL-6),STAT3,and transient receptor potential vanilloid-1 channel(TRPV1)were detected by Western Blot.Results Compared with the normal group,rats in the model group showed obvious asthma symptoms,severe inflammatory cell infiltration was seen in the lung tissue,bronchial epithelial cell necrosis,ciliated adhesion,mucus,and RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,TRPV1 were elevated(P＜0.05);compared with the model group,rats in the low-and high-dose groups of Maxing Shigan Decoction showed significant improvement in asthma symptoms,reduction in lung and bronchial injury,and dose-dependent reduction in RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,and TRPV1(P＜0.05);compared with the high-dose group of Maxing Shigan Decoction,the rats in the high-dose Maxing Shigan Decoction+Col group showed increased asthma,increased lung and bronchial injury,and increased RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,and TRPV1(P＜0.05).Conclusion Maxing Shigan Decoction can effectively improve cough variant asthma in rats,and its mechanism is related to the inhibition of IL-6/STAT3 signaling pathway and the high expression of TRPV1.

The effect of sinomenine on proliferation and apoptosis of multiple myeloma U266 cells through regulation of STAT3 and NF-κB signaling pathways / 安徽医科大学学报

Objective @#To investigate the effect of sinomenine on proliferation and migration of multiple myeloma (MM) cells by regulating STAT3 and NF-κB signaling pathway．@*Methods @# The cultured U266 cells were treated with different concentrations of sinomenine (0，0．5，1，2 mmol / L) ．The control group was added DMSO with 0．5% concentration．CCK-8 assay was used to detect the proliferation of U266 cells．Flow cytometry was used to detect the apoptosis of U266 cells．Western blot assay was used to detect the expression levels of apoptosis-related proteins， STAT3 and NF-κB signaling pathway proteins in the each group. @*Results @# Compared with CON group，the apopto- sis of U266 cells increased after Sinomenine treatment，the proliferation was inhibited ; B lymphoma-2 (Bcl-2) mye- loid and cell leukemia-1 (Mcl-1) expression level decreased ; activated Caspase-3 (cleaved Caspase-3) and PARP (Cleaved Caspase-3) expression levels increased ; the activity of STAT3 and NF-κB signaling pathway decreased. @*Conclusion @# Sinomenine can down-regulate the activity of STAT3 and NF-κB signaling pathway，promote the apop- tosis of U266 cells and inhibit the proliferation of U266 cells.

Paeonol ameliorates hepatic inflammation and oxidative stress injury on murine with alcoholic liver injury by regulating JAK2/STAT3 signaling pathway / 中国药理学通报

Aim To investigate the mechanism of action of paeonol based on JAK2/STAT3 signaling pathway to ameliorate liver inflammation and oxidative stress injury induced by acute alcohol stimulation in mice. Methods C57BL/6 mice were randomly divided into normal group, model group, silibinin group (36. 8 mg • kg

Mechanism of Modified Danggui Shaoyao San in treatment of chronic atrophic gastritis based on network pharmacology and experimental verification / 中国药理学通报

Aim To predict the targets of Modified Danggui Shaoyao San ( MDSS) in the treatment of chronic atrophic gastritis ( CAG) based on network pharmacology and vertify the results based on experim-ention. Methods TCMSP, SWISS TARGETS, GENE CARDS and OMIM databases were used to screen the therapeutic targets of MDSS for CAG. STRING database and Cytoscape software were used to construct the protein interaction network and screen the core targets. Metascape database was used for GO analysis and KEGG enrichment pathways. And molecular docking was used for target validation. CAG rat model was pre¬pared by N-methyl-N'-nitroso-N-nitroguanidine free drink combined with sodium salicylate gastric lavage. The pathology of rat gastric mucosa was observed by hematoxylin-eosin staining,and the ultrastructure of ep¬ithelial cells was observed by transmission electron mi-croscopy. The serum IL-6 and IL-10 content was detected by enzyme-linked immunosorbent assay, and the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in rats was detected by qPCR and Western blot. Results MDSS acted on 189 targets, mainly involved in response to oxidative stress and apoptotic signaling pathway. KEGG analysis related to pathways in cancer and JAK-STAT signaling pathway. The experimental results showed that the MDSS could improve the degree of atrophy of gastric mucosa in CAG rats and improve the status of epithelial cells, down-regulate the serum IL-6 content of CAG rats, up-regulate the IL-10 content, and reduce the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in gastric mucosa with statistical significance. Conclusions MDSS treats CAG through multiple active ingredients, targets, and pathways, the mechanism of which may be related to the inhibition of the JAK2/STAT3 signaling pathway.

Mechanisms of Euphorbia fischeriana steud. alcohol extracts intreatment of HCC based on network pharmacology and experiment verification / 中国药理学通报

Aim To investigate the molecular mechanisms of alcohol extracts of Euphorbia fischeriana steud. against hepatocellular carcinoma (HCC) through a combination of network pharmacology analysis and experimental validation. Methods The active ingredients and targets of alcohol extracts of Euphorbia fischeriana steud. were determined through TCMSP, Swiss ADME, Swiss Target Prediction database and references. The databases DisGeNET and GeneCards were employed to screen potential HCC-related genes. Venny platform, STRING platform and Cytoscape software were applied to construct active ingredient-target-disease and protein-protein interaction (PPI) network maps. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed using the DAVID database. To assess the effects of Euphorbia fischeriana steud. alcohol extracts on BEL-7402 cells, the proliferation and apoptosis were detected by CCK-8, EdU and flow cytometry assays, and the related protein levels of JAK2/STAT3 pathway were analyzed by Western blot. Additionally, H22 hepatocellular carcinoma mouse model was used to evaluate the in vivo efficacy of Euphorbia fischeriana steud. alcohol extracts. Results A total of 916 HCC targeted genes, 30 active ingredients containing the related 567 potential targeted genes, and 115 intersection targets of disease and compounds were obtained. KEGG enrichment analysis identified JAK2/STAT3 signaling as a critical pathway. In vitro experiments showed the alcohol extracts of Euphorbia fischeriana steud. could inhibit proliferation, promote apoptosis and suppress JAK2/STAT3 signaling pathway in a dose-dependent manner in BEL-7402 cells. In addition, the alcohol extracts of Euphorbia fischeriana steud., either alone or in combination with sorafenib, dramatically blocked tumor growth in in vivo tests. Conclusions Euphorbia fischeriana steud. alcohol extracts have anti-cancer effects in HCC, and the molecular mechanisms may be connected to the regulation of JAK2/STAT3 signaling pathway.

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway / 中国中药杂志

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

Effect of Scutellarin on Proliferation of Acute Myeloid Leukemia Cells and Its Related Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of scutellarin (SCU) on proliferation, cell cycle and apoptosis of acute myeloid leukemia (AML) cells and its related molecular mechanism.@*METHODS@#Human AML HL-60 cells were cultured in vitro. The cells were treated with SCU at the concentration of 0, 2, 4, 8, 16, 32, 64 μmol/L, and the inhibition rate of cell proliferation was detected by CCK-8 method. Then HL-60 cells were treated with SCU at the concentration of 4, 8, 16 μmol/L, and the negative control group (NC group) was set. The cell cycle distribution and apoptosis were detected by flow cytometry, and the expression of cell cycle, apoptosis and JAK2/STAT3 pathway related proteins were detected by Western blot.@*RESULTS@#SCU significantly inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner(r =0.958,r =0.971). Compared with NC group, the proportion of cells in G0/G1 phase and apoptosis rate of HL-60 cells in 4, 8, 16 μmol/L SCU group were significantly increased, and the proportion of cells in S phase was significantly decreased (P <0.05). The relative protein expression levels of p21, p53, caspase-3 and Bax were significantly increased, while the relative protein expression levels of CDK2, cyclin E and Bcl-2 were significantly decreased (P <0.05). The ratio of p-JAK2/JAK2 and p-STAT3/STAT3 were significantly decreased (P <0.05). The changes of above-mentioned indexes were concentration dependent.@*CONCLUSION@#SCU can inhibit the proliferation of AML cells, induce cell cycle arrest and apoptosis, and its mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

Chemokine CXCR4 regulates gastric cancer development through IL-6/STAT3 signaling pathway / 免疫学杂志

The objective of this study is to investigate the effect of chemokine CXCR4 on the apoptosis of gastric cancer cells through IL-6/STAT3 signaling pathway and to explore the related mechanisms.Fluorescence quantitative PCR and Western blot were performed to detect the mRNA and protein expression levels of the chemokine CXCR4 in human gastric cancer tissues and adjacent non-cancerous tissues(PCT).Next,CXCR4 knockdown and overexpression were achieved by transfecting SGC7901 gastric cancer cell line with lentiviral vectors.TUNEL staining was used to evaluate the apoptosis of SGC7901 cells,while MTT assay was employed to measure cell proliferation.Western blot was conducted to determine the expression of apoptosis-related proteins Bax and Bcl2.Further,enzyme-linked immunosorbent assay(ELISA)was employed to measure the secretion levels of inflammatory cytokines.Real-time fluorescent quantitative PCR(RT-PCR)was utilized to quantify the expression of IL-6 mRNA in the IL-6/STAT3 signaling pathway,and Western blot was performed to analyze the expression of STAT3-Ser727 protein.In addition,after knocking down CXCR4 in SGC7901 cells,IL-6/STAT3 signaling pathway agonist lipopolysaccharide(LPS)was transfected,while in CXCR4-overexpressing SGC7901 cells,IL-6/STAT3 signaling pathway inhibitors angoline or bruceantinol were transfected.Then TUNEL staining was used to assess cell apoptosis,and Western blotting was performed to examine the expression levels of apoptosis-related proteins Bax and Bcl2 in these cells.Data showed that the expression of immune chemokine CXCR4 was increased in gastric cancer tissues,as compared with adjacent non-cancerous tissues.Single-cell gel electrophoresis analysis indicated that knockdown or overexpression of CXCR4 do not induce DNA damage in SGC7901 cells.TUNEL staining,MTT cell proliferation assay and Western blotting demonstrated that knockdown of CXCR4 in SGC7901 cells promoted the apoptosis in SGC7901 cells,while overexpression of CXCR4 inhibited the apoptosis.ELISA showed that knockdown of CXCR4 in SGC7901 cells promotes the expression of pro-inflammatory factors IL-1β and TNF-α,while inhibited the expression of anti-inflammatory factors IL-10 and TGF-β.Conversely,overexpression of CXCR4 demonstrated opposite effects.Finally,the activation of the IL-6/STAT3 signaling pathway significantly reduced the apoptosis induced by knocking down CXCR4 in iSGC7901 cells,whereas the inhibition of IL-6/STAT3 signaling pathway can significantly suppressed the induction of SGC7901 cells proliferation induced by CXCR4 overexpress.In conclusion,immunochemokine CXCR4 regulates gastric cancer cell apoptosis and inflammatory cytokines secretion through IL-6/STAT3 signaling pathway.

Shaoyaotang Alleviates Ulcerative Colitis by Regulating Th17/Treg Balance Through IL-6/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the role of interleukin-6 （IL-6）/signal transducer and activator of transcription 3 （STAT3） pathway in the balance of T helper 17 （Th17）/regulatory T （Treg） cells in ulcerative colitis （UC） with internal dampness-heat accumulation syndrome and the intervention mechanism of Shaoyaotang. MethodA total of 60 SD rats were randomized into blank group （equivalent volume of normal saline）， model group （equivalent volume of normal saline）， western medicine control group （0.42 g·kg-1 mesalazine）， and low-dose （11.1 g·kg-1）， medium-dose （22.2 g·kg-1）， and high-dose （44.4 g·kg-1） Shaoyaotang groups. UC with internal dampness-heat accumulation syndrome was induced in rats with the compound method except for the blank group. The administration lasted 14 days for each group. At 24 h after the last administration， rats were killed and the spleen and colon tissues were separated. The histopathological changes of colon were observed based on hematoxylin and eosin （HE） staining and the levels of interleukin-17 （IL-17） and transforming growth factor-β1 （TGF-β1） in colon tissue were detected by immunohistochemistry （IHC）. Flow cytometry was employed to determine the levels of Th17/Treg cells in the spleen， and Western blot to measure the levels of IL-6 and STAT3 proteins in colon tissue. ResultCompared with the blank group， the model group had lesions such as congestion and erosion， low percentage of spleen Treg cells （P<0.01）， high percentage of Th17 cells （P<0.01）， and high levels of IL-6 and STAT3 proteins in colon tissue （P<0.01）. Compared with the model group， the administration groups showed alleviation of colon injury， high percentage of spleen Treg cells （P<0.05， P<0.01）， low percentage of Th17 cells （P<0.01）， and low levels of IL-6 and STAT3 proteins in colon tissue （P<0.01）. ConclusionShaoyaotang regulates the balance of Th17/Treg by inhibiting the IL-6/STAT3 pathway， thereby relieving the pathological damage of UC rats with internal dampness-heat accumulation syndrome and affecting their immune function.

Inhibitory effect and molecular mechanism of sinomenine on human hepatocellular carcinoma HepG2 and SK-HEP-1 cells / 中国中药杂志

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.

Influence of Limonin on Malignant Biological Behavior of Non-small Cell Lung Cancer Cells by Regulating JAK2/STAT3 Signaling Pathway / 肿瘤防治研究

Objective To investigate the influence of limonin on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells by regulating the protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods CCK-8 method was applied to detect the survival rate of A549 cells treated with different concentrations of limonin (0, 5, 10, 25, 50, 75, 100 μmol/L). A549 cells were separated into normal culture (NC) group, low-dose limonin group (treatment with 10 μmol/L limonin for 24 h), medium-dose limonin group (treatment with 25 μmol/L limonin for 24 h), high-dose limonin group (treatment with 50 μmol/L limonin for 24 h), coumermycin A1 group (treatment with 10 μmol/L JAK2 activator coumermycin A1+50 μmol/L limonin for 24 h), and AG490 group (treatment with 10 μmol/L JAK2 inhibitor AG490+50 μmol/L limonin for 24 h). Clone formation assay was applied to detect the clones of each group of cells. Transwell assay was applied to detect cell migration and invasion, and flow cytometry was applied to detect apoptosis. Western blot analysis was applied to detect the protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, E-cadherin, N-cadherin, and vimentin in each group. Results The viability of A549 cells decreased significantly in a limonin concentration-dependent manner (P < 0.05), with IC50 of 45.16±1.66 μmol/L. Concentrations of 10, 25, and 50 μmol/L were selected for subsequent experiments. The numbers of clones, migration, and invasion of A549 cells and the protein expression levels of IL-6, p-JAK2, p-STAT3, N-cadherin, and vimentin in the low-, medium-, and high-dose limonin groups significantly decreased, compared with those in the NC group, and the apoptosis rate and E-cadherin protein expression significantly increased (P < 0.05). The JAK2 activator coumermycin A1 attenuated the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and attenuated the apoptosis ability. The JAK2 inhibitor AG490 enhanced the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and enhanced the apoptosis ability. Conclusion Limonin can inhibit the malignant biological behavior of NSCLC cells, such as proliferation, migration, and invasion, by inhibiting the JAK2/STAT3 pathway.

AG490 regulates JAK2/STAT3 pathway in M2-like macrophages via promoting the proliferation of gastric cancer cells and inhibiting apoptosis / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of JAK2/STAT3 signaling pathway inhibitor AG490 on the functional phenotype of M2-like macrophages and its effects on proliferation, apoptosis, migration, and invasion of gastric cancer cells. 【Methods】 Human mononuclear cell line THP-1 was induced to differentiate into M0 macrophages by PMA in vitro. The M1-like phenotype was induced by LPS and IFN-γ, and M2-like phenotype was induced by IL-4 and IL-13, respectively, and identified by immunofluorescence labeling CD68, CD86 and CD206. The mRNA expressions of CD163, Arg1, CCL22, PPARγ, IL-10, IL-20 and TNF-α were determined by RT-qPCR. The expressions of key proteins in JAK2/STAT3 signaling pathway were detected by Western blotting. M2-like macrophages were treated with JAK2/STAT3 inhibitor (AG490) to observe the expression level of marker genes for M2 like phenotype. Macrophages were co-cultured with gastric cancer cells, and the effects of the macrophages on proliferation, migration, invasion, and apoptosis of gastric cancer cells were detected by CCK-8 method, healing assay, transwell intracellular Matrigel invasion assay, and flow cytometry. The xenograft tumor model of MKN45 gastric cancer in nude mice was prepared, and the tumor size and quality were observed for 20 days after the model was established. 【Results】 THP-1 cells were induced into M1-like macrophages and M2-like macrophages. M1-like marker (CD86) and M2-like marker (CD206) were identified by flow cytometry. The P38MAPK, JAK2, p-STAT3/STAT3 protein levels of M2-like macrophages treated with AG490 were significantly reduced. The mRNA expression levels of Arg1, CCL22, PPARγ and IL-10 were significantly reduced in the group of M2-like macrophages treated with AG490. Co-culture of M2-like macrophages with gastric cancer cells could promote gastric cancer cell viability, increase migration and invasion ability, and inhibit apoptosis. When the group of M2-like macrophages treated with AG490 was co-cultured with gastric cancer cells, the proliferation activity of MKN-45 cells and MGC823 was significantly lower than that in M2 group (1.047±0.062 vs. 1.426±0.076, 1.149±0.006 vs. 1.301±0.015). Compared to M2 group, the migration (100.0%±5.73% vs. 72%±3.85%) and invasion ability (100.0%±7.40% vs. 60%±6.54%) of MGC823 gastric cancer cells in AG490 treatment group were significantly reduced. The apoptosis rate of MGC823 cells in the AG490 treated group was significantly higher than that in M2 group (27.51%±0.70% vs. 20.82%±0.92%). In the nude mouse xenograft tumor model, the volume and weight of the transplanted tumor collected at day 20 were significantly lower in AG490 treated group than in M2 group (736.04±182.34 vs. 1 080.5±250.57)mm3, (0.64±0.11 vs. 0.87±0.17)g. 【Conclusion】 AG490 downregulates the activation level of JAK2/STAT3 signaling pathway in M2-like macrophages, inhibits M2-type polarization, partially reverses the cancer-promoting function of M2-like macrophages, inhibits proliferation, migration and invasion of gastric cancer cells, and induces apoptosis. The JAK2/STAT3 signaling pathway can be further studied as a potential therapeutic target for gastric cancer.

Cinnamaldehyde inhibits pancreatic cancer cell proliferation and sternness via modulation of CD44s/STAT3 signaling pathway / 中国药理学通报

Aim To explore the effects of cinnamaldehyde on the proliferation and stemness of pancreatic cancer PANC-1 cells, and its possible mechanism of action.Methods CCK8 assay was used to detect the effect of cinnamaldehyde treatment on cell viability at different concentrations(0, 5, 15, 20, 30, 50, 70, 100, 150 μmol·L-1)and different intervention time(24, 48, 72 h).CFSE proliferation assay was used to detect the inhibitory effects of cinnamaldehyde on PANC-1 cells.Colony formation assay was employed to determine the colony-forming ability of PANC-1 cells after cinnamaldehyde treatment.The sphere formation assay was employed to detect the effects of cinnamaldehyde on sphere-forming ability in PANC-1 cells.Western blot analysis and qRT-PCR analysis were applied to determine the expression levels of Nanog, Sox-2 and Oct-4.Flow cytometry was used to detect the percentage of CD44+CD24+ cells and ALDH+ cells in cinnamaldehyde treated and untreated PANC-1 cells.Western blot analysis was used to detect the effects of cinnamaldehyde on the expression of CD44s, p-STAT3 and STAT3 in PANC-1 cells.Results Cinnamaldehyde suppressed cell viability in a dose- and time-dependent manner, and inhibited tumor-cell proliferation and colony forming ability significantly in a dose-dependent manner in PANC-1 cells.Sphere-forming assay showed that cinnamaldehyde could significantly inhibit sphere-forming ability in suspension culture of PANC-1 cells.The mRNA and protein expression levels of three stemness-related genes were down-regulated after cinnamaldehyde treatment.In addition, cinnamaldehyde treatment significantly decreased the proportion of CD44+CD24+ cells and ALDH+ cells.Western blotting showed that cinnamaldehyde inhibited the expression of CD44s and p-STAT3, while it had no effect on the expression of STAT3.With the addition of STAT3 activator(Colivelin TFA), the inhibition of cinnamaldehyde on proliferation and tumor-cell stemness in PANC-1 cells was partially rescued.Conclusions Cinnamaldehyde significantly inhibits the proliferation and tumor-cell stemness of pancreatic cancer PANC-1 cells, and the mechanism could be related to the modulation of CD44s/STAT3 signaling pathway.

Knocking-down Synaptotagmin 1 Attenuated Colitis via Suppressing the IL-6/STAT3 Signaling Pathway / 中国生物化学与分子生物学报

Synaptotagmin 1 (Sytl) is a member of the Synaptotagmin family and plays a role in neurotransmitter vesicle transport and exoeytosis. It has been reported that Sytl appears to be expressed in the intestinal epithelium, but the biological function of Sytl in colitis remains poorly understood. This study aimed to investigate the effects of Sytl in the inflammatory response and intestinal epithelial regeneration in colitis using Sytl transgenic mice and dextran sulfate sodium (DSS)-induced ulcerative colitis mode. qRT-PCR and Western blotting were employed to analyze the dynamic changes of Sytl in colitis. H&E staining, immunostaining and Western blotting were used to explore the roles of Sytl in the inflammatory response and in the regeneration and repair of intestinal epithelium in colitis. The results showed that the expression level of Sytl was indeed high in the colonic epithelium of wild-type mice and the intestinal epithelial cells of the adjacent tissues of colorectal cancer patients. Consistently, DSS-induced inflammation progressively resulted in marked upregulation of Sytl in the colon (P<0.01). In DSS-induced colitis, both the body weight loss and colonic shortening were dampened in Sytl loss-of-function mice compared with the control group (P < 0.05), while the number of regenerated crypts and Ki67 proliferating cells were also increased (P<0.01). Additionally, there were less infiltration of CD45 immune cells and F 4/80 macrophages and the expression of pro-inflammatory cytokines IL-6, TNFα and I L l-β, which were related with the severity of inflammation in the inflammatory bowel disease (I B D), were significantly decreased after Sytl deletion (P<0.05). Immunohistochemistry staining and Western blotting results further showed that IL-6 and p-STAT3 was significantly downregulated in Sytl knockdown mice (P<0.05). Taken together, these results suggested that knocking-down of Sytl may improve colitis by inhibiting the IL6/STAT3 signaling pathway.