Lung-derived exosomes regulate the function of mesenchymal stem cells and alleviate phosgene-induced lung injury via miR-34c-3p.

Phosgene may induce acute lung injury (ALI) when a person is exposed to it. Mesenchymal stem cells (MSCs) were affirmed to have therapeutic effects on phosgene-induced ALI. In a previous study, ALI exosomes have been confirmed to promote the proliferation and migration of MSCs. However, the mechanism of this phenomenon is still unclear. MicroRNAs (miRNAs) are essential in the physiological process of cells. In this study, lung-derived exosomes were isolated from phosgene-exposed and normal rats, respectively, through ultracentrifugation and cultured MSCs with these exosomes. We found that rno-miR-34c-3p was downregulated in MSCs cocultured with ALI exosomes. MiR-34c-3p inhibitor promoted the proliferation and migration of MSCs. Moreover, the dual-luciferase reporter assay demonstrated that miR-34c-3p regulated Janus kinase 1 (JAK1) expression. The miR-34c-3p inhibitor also significantly activated the JAK1/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In conclusion, ALI exosomes decrease the miR-34c-3p expression levels, influencing MSCs via the JAK1/STAT3 signaling pathway.

miR-106b Promotes Metastasis of Early Gastric Cancer by Targeting ALEX1 in Vitro and in Vivo.

BACKGROUND/AIMS: Aberrant expression of miR-106b is a specific symptom of many solid carcinomas. Overexpression of miR-106b has been observed in gastric cancer. The effect of miR-106b on gastric cancer has been investigated in different cell culture models. However, the effect of miR-106b on metastasis of early gastric cancer (EGC) remains unknown. METHODS: In the study, qRT-PCR, FISH, western blot, luciferase reporter assay, migration and invasion assays, flow cytometry and TUNEL staining were used to investigate the effect of miR-106b on metastasis of EGC. RESULTS: To explore the function of miR-106b in EGC, we investigated the downstream signaling of miR-106b and found that ALEX1 was a direct target of miR-106 in gastric cancer cells. Up-regulation of ALEX1 effectively rescued the cell apoptosis induced by miR-106b inhibitor and promoted the expression levels of phosphorylation of JAK1 and STAT3. Moreover, overexpression of JAK1 reduced the cell apoptosis induced by miR-106b inhibitor and decreased the expression levels of the apoptotic proteins in gastric cancer cells. Furthermore, down-regulation of miR-106b promoted apoptosis of gastric cancer cells via inhibiting JAK1/STAT3 signaling pathway in vitro and in vivo. In addition, GLPG0643, a JAK1 inhibitor, enhanced the inhibitory effect of miR-106b inhibitor on gastric cancer growth in vivo. CONCLUSION: These findings provided a potential therapeutic manner for the treatment of metastasis of EGC in clinic.

Sinapine thiocyanate alleviates intervertebral disc degeneration by not regulating JAK1/STAT3/NLRP3 signal pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is a major cause of low back pain. Sinapine thiocyanate (ST) has been reported to have a wide range of biological activities. However, the treatment of IDD with ST has not been studied. OBJECTIVES: To explore the role and mechanism of ST treatment in IDD. MATERIAL AND METHODS: Nucleus pulposus cells (NPCs) were induced using lipopolysaccharide (LPS), which was used as an in vitro model of IDD. Cell activity, oxidative stress-related indicators and protein expression were detected using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, enzyme-linked immunosorbent assay (ELISA) and western blot. Pyroptosis was evaluated with propidium iodide (PI)/Hoechst double staining and immunofluorescence for NOD-like receptor protein 3 (NLRP3), and pyroptosis-related proteins and inflammatory factors were measured with western blot and ELISA. The pathological changes of IDD were assessed with hematoxylin & eosin (H&E) and safranin-O staining. RESULTS: Our results showed that ST alleviated LPS-induced degeneration of NPCs, as evidenced by reducing reactive oxygen species (ROS), malondialdehyde (MDA), matrix metalloproteinase-13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and increasing collagen II and aggrecan expression. Moreover, ST repressed LPS-induced pyroptosis by inhibiting NLRP3, caspase-1 p20, interleukin (IL)-1ß and IL-18. Further studies showed that ST did not restrain the activation of the JAK1/STAT3 signaling pathway induced by colivelin, or of the enhanced pyroptosis induced by polyphyllin VI. Sinapine thiocyanate alleviated IDD in vivo and suppressed NLRP3-mediated pyroptosis and the JAK1/STAT3 signaling pathway. CONCLUSIONS: Sinapine thiocyanate could alleviate IDD, although this did not include a reduction in NLRP3-mediated pyroptosis and inactivation of the JAK1/STAT3 signaling pathway, thus potentially being a candidate drug for IDD treatment.

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signaling pathway.

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) remain poorly understood. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of Gpr160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Consistent with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Graveoline attenuates D-GalN/LPS-induced acute liver injury via inhibition of JAK1/STAT3 signaling pathway.

Graveoline exhibits various biological activities. However, only limited studies have focused on its hepatoprotective properties. This study evaluated the anti-inflammatory and hepatoprotective activities of graveoline, a minor 2-phenylquinolin-4-one alkaloid isolated from Ruta graveolens L., in a liver injury model in vitro and in vivo. A network pharmacology approach was used to investigate the potential signaling pathway associated with the hepatoprotective activity of graveoline. Subsequently, biological experiments were conducted to validate the findings. Topological analysis of the KEGG pathway enrichment revealed that graveoline mediates its hepatoprotective activity through genes associated with the hepatitis B viral infection pathway. Biological experiments demonstrated that graveoline effectively reduced the levels of alanine transaminase and aspartate transaminase in lipopolysaccharide (LPS)-induced HepG2 cells. Graveoline exerted antihepatitic activity by inhibiting the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and elevated the anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) in vitro and in vivo. Additionally, graveoline exerted its hepatoprotective activity by inhibiting JAK1 and STAT3 phosphorylation both in vitro and in vivo. In summary, graveoline can attenuate acute liver injury by inhibiting the TNF-α inflammasome, activating IL-4 and IL-10, and suppressing the JAK1/STAT3 signaling pathway. This study sheds light on the potential of graveoline as a promising therapeutic agent for treating liver injury.

The leukemia inhibitory factor regulates fibroblast growth factor receptor 4 transcription in gastric cancer.

PURPOSE: The gastric adenocarcinoma (GC) represents the third cause of cancer-related mortality worldwide, and available therapeutic options remain sub-optimal. The Fibroblast growth factor receptors (FGFRs) are oncogenic transmembrane tyrosine kinase receptors. FGFR inhibitors have been approved for the treatment of various cancers and a STAT3-dependent regulation of FGFR4 has been documented in the H.pylori infected intestinal GC. Therefore, the modulation of FGFR4 might be useful for the treatment of GC. METHODS: To investigate wich factors could modulate FGFR4 signalling in GC, we employed RNA-seq analysis on GC patients biopsies, human patients derived organoids (PDOs) and cancer cell lines. RESULTS: We report that FGFR4 expression/function is regulated by the leukemia inhibitory factor (LIF) an IL-6 related oncogenic cytokine, in JAK1/STAT3 dependent manner. The transcriptomic analysis revealed a direct correlation between the expression of LIFR and FGFR4 in the tissue of an exploratory cohort of 31 GC and confirmed these findings by two external validation cohorts of GC. A LIFR inhibitor (LIR-201) abrogates STAT3 phosphorylation induced by LIF as well as recruitment of pSTAT3 to the promoter of FGFR4. Furthermore, inhibition of FGFR4 by roblitinib or siRNA abrogates STAT3 phosphorylation and oncogentic effects of LIF in GC cells, indicating that FGFR4 is a downstream target of LIF/LIFR complex. Treating cells with LIR-201 abrogates oncogenic potential of FGF19, the physiological ligand of FGFR4. CONCLUSIONS: Together these data unreveal a previously unregnized regulatory mechanism of FGFR4 by LIF/LIFR and demonstrate that LIF and FGF19 converge on the regulation of oncogenic STAT3 in GC cells.

Lipophilic Constituents in Salvia miltiorrhiza Inhibit Activation of the Hepatic Stellate Cells by Suppressing the JAK1/STAT3 Signaling Pathway: A Network Pharmacology Study and Experimental Validation.

Liver fibrosis is currently a global health challenge with no approved therapy, with the activation of hepatic stellate cells being a principal factor. Lipophilic constituents in Salvia miltiorrhiza (LS) have been reported to improve liver function and reduce the indicators of liver fibrosis for patients with chronic hepatitis B induced hepatic fibrosis. However, the pharmacological mechanisms of LS on liver fibrosis have not been clarified. In this study, 71 active compounds, 342 potential target proteins and 22 signaling pathways of LS were identified through a network pharmacology strategy. Through text mining and data analysis, the JAK1/STAT3 signaling pathway was representatively selected for further experimental validation. We firstly confirmed the protective effect of LS on liver fibrosis in vivo by animal experiments. Hepatic stellate cells, which proliferated and displayed a fibroblast-like morphology similar to activated primary stellate cells, were applied to evaluate its underlying mechanisms. The results showed that LS could inhibit the cell viability, promote the cell apoptosis, decrease the expression of liver fibrosis markers, and downregulate the JAK1/STAT3 signaling pathway. These results demonstrated that LS could exert anti-liver-fibrosis effects by inhibiting the activation of HSCs and regulating the JAK1/STAT3 signaling pathway, which is expected to benefit its clinical application.

Reversal of alopecia areata, osteoporosis follow treatment with activation of Tgr5 in mice.

BACKGROUND: Alopecia areata is an autoimmune hair loss disease with infiltration of pro-inflammatory cells into hair follicles. The role of Tgr5 in dermatitis has attracted considerable attention. The present study aimed to investigate the effect of Tgr5 in the development of Alopecia areata. METHODS: The study utilized a comparison control group design with four groups of wild-type group, wild-type+INT777 group, Tgr5-/- group, and Tgr5-/-+INT777 group. The mice were treated with INT777 (30 mg/kg/day) or the carrier solution (DMSO) intraperitoneally for 7 weeks, and the back skin was collected and analyzed by histology and immunohistochemistry staining. The lumbar vertebrae 4 has also been analyzed by DXA and Micro-CT. RESULTS: Tgr5-/- mice displayed the decreasingly significant in hair area and length, skin thickness, and the ratio of anagen and telogen, collagen, and mast cell number and loss the bone mass than WT group. After treating with INT777, the appearance of alopecia areata and bone microstructure has improved. Immunohistochemistry and qPCR analysis showed that activation of Tgr5 can down-regulate the express of JAK1, STAT3, IL-6, TNF-α, and VEGF. CONCLUSION: These findings indicate that activation of Tgr5 mediated amelioration of alopecia areata and osteoporosis by down-regulated JAK1-STAT3 signaling pathway.

Long non-coding RNA SNHG16, binding with miR-106b-5p, promoted cell apoptosis and inflammation in allergic rhinitis by up-regulating leukemia inhibitory factor to activate the JAK1/STAT3 signaling pathway.

Allergic rhinitis (AR) is a type I hypersensitive disease. Long non-coding RNA (lncRNA) SNHG16 acts as an oncogene in a variety of tumors and promotes the occurrence of inflammation in many inflammatory diseases. The study aims to investigate the expression of SNHG16 and its potential biological functions in AR. RT-qPCR results showed that the expression of SNHG16 in AR was up-regulated. The AR cell model was constructed by stimulating primary nasal mucosal epithelial cells from AR patients with IL-13. After knocking down the expression of lncRNA SNHG16, cell apoptosis was detected by flow cytometry, and the expression of inflammatory factors was detected by ELISA. The results showed that SNHG16 promoted cell apoptosis and inflammation. Then, bioinformatics analysis was used to screen miRNAs bound with SNHG16. Luciferase reporter gene assay and RNA pull-down experiment were used to verify the relationship. We found that the expression of miR-106b-5p was down-regulated and leukemia inhibitory factor (LIF) expression was up-regulated in the AR cell model. The expression of phospho-Janus kinase 1 and p-signal transducer and activator of transcription 3 (STAT3) were detected by Western blotting. Silencing the expression of LIF could inhibit the activity of JAK1/STAT3 pathway and further inhibit cell apoptosis and the occurrence of inflammation. Then transfected SNHG16 shRNA alone or together with miR-106b-5p antagomir into the AR cell model, we found that silencing the expression of SNHG16 down-regulated the expression of LIF and inhibited the activity of the JAK1/STAT3 pathway, cell apoptosis, and inflammation. However, miR-106b-5p antagomir weakened its inhibitory effects. The role of SNHG16 in AR was further verified by the ovalbumin-induced AR mouse model in vivo. In conclusion, SNHG16 up-regulates LIF expression by binding with miR-106b-5p, thus promoting the activity of JAK1/STAT3 pathway, and promoting the development of AR. These results provide new targets for the treatment of AR and may help reduce the damage caused by AR.

HDAC3 Downregulation Improves Cerebral Ischemic Injury via Regulation of the SDC1-Dependent JAK1/STAT3 Signaling Pathway Through miR-19a Upregulation.

Histone deacetylase (HDAC) inhibitors can protect the brain from ischemic injury. This study aimed to identify the regulation of HDAC3 in cerebral ischemic injury. Middle cerebral artery occlusion (MCAO) was performed to establish a mouse model with cerebral ischemic injury, in which expression of HDAC3 and miR-19a was evaluated using RT-qPCR. In MCAO mice with silencing of HDAC3, infarct volume was determined using 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and serum levels of TNF-α, IL-6, and IL-8 were measured using ELISA. An in vitro model was constructed in human umbilical vein endothelial cells (HUVECs) with oxygen-glucose deprivation/reoxygenation (OGD/R), followed by gain- and loss-of-function experiments. Relationships among miR-19a, HDAC3, and syndecan-1 (SDC1) were explored using RIP, ChIP, and dual-luciferase reporter assays. The expression of HDAC3, SDC1, JAK1, and STAT3 along with the extent of JAK1 and STAT3 phosphorylation was measured by Western blot analysis. HUVEC viability, apoptosis, and angiogenesis were assessed by CCK-8, flow cytometry, and angiogenesis assays in vitro separately. We found elevated HDAC3 and downregulated miR-19a expression in the MCAO mice. Decreased TNF-α, IL-6, and IL-8 serum levels were observed in response to silencing of HDAC3. HDAC3 inhibited the expression of miR-19a, which in turn targeted SDC1, leading to JAK1/STAT3 signaling pathway activation. HDAC3 overexpression or miR-19a inhibition repressed HUVEC viability and angiogenesis but enhanced HUVEC apoptosis. Our data unraveled the mechanism whereby HDAC3 inhibition ameliorated cerebral ischemic injury by activating the JAK1/STAT3 signaling pathway through miR-19a-mediated SDC1 inhibition.

[Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells].

OBJECTIVE: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. METHODS: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 µg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 µg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. RESULTS: Calenduloside E below 20 µg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1ß, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. CONCLUSIONS: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.