Dapagliflozin exerts anti-apoptotic effects by mitigating macrophage polarization via modulation of the phosphoinositide 3-kinase/protein kinase B signaling pathway

BACKGROUND Macrophages are central to the orchestration of immune responses,inflammatory processes,and the pathogenesis of diabetic complications.The dynamic polarization of macrophages into M1 and M2 phenotypes critically modulates inflammation and contributes to the progression of diabetic nephropathy.Sodiumglucose cotransporter 2 inhibitors such as dapagliflozin,which are acclaimed for their efficacy in diabetes management,may influence macrophage polarization,thereby ameliorating diabetic nephropathy.This investigation delves into these mechanistic pathways,aiming to elucidate novel therapeutic strategies for diabetes.AIM To investigate the inhibitory effect of dapagliflozin on macrophage M1 polarization and apoptosis and to explore its mechanism of action.METHODS We established a murine model of type 2 diabetes mellitus and harvested peritoneal macrophages following treatment with dapagliflozin.Concurrently,the human monocyte cell line cells were used for in vitro studies.Macrophage viability was assessed in a cell counting kit 8 assay,whereas apoptosis was evaluated by Annexin V/propidium iodide staining.Protein expression was examined through western blotting,and the expression levels of macrophage M1 surface immunosorbent assay,and quantitative real-time polymerase chain reaction analyses.RESULTS Dapagliflozin attenuated M1 macrophage polarization and mitigated apoptosis in the abdominal macrophages of diabetic mice,evidenced by the downregulation of proapoptotic genes(Caspase 3),inflammatory cytokines[interleukin(IL)-6,tumor necrosis factor-α,and IL-1β],and M1 surface markers(inducible nitric oxide synthase,and cluster of differentiation 86),as well as the upregulation of the antiapoptotic gene BCL2.Moreover,dapagliflozin suppressed the expression of proteins associated with the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway(PI3K,AKT,phosphorylated protein kinase B).These observations were corroborated in vitro,where we found that the modulatory effects of dapagliflozin were abrogated by 740Y-P,an activator of the PI3K/AKT signaling pathway.CONCLUSION Dapagliflozin attenuates the polarization of macrophages toward the M1 phenotype,thereby mitigating inflammation and promoting macrophage apoptosis.These effects are likely mediated through the inhibition of the PI3K/AKT signaling pathway.

Mesalazine alleviated the symptoms of spontaneous colitis in interleukin-10 knockout mice by regulating the STAT3/NF-κB signaling pathway

BACKGROUND Excessive endoplasmic reticulum(ER)stress in intestinal epithelial cells can lead to damage to the intestinal mucosal barrier,activate the signal transducer and activator of transcription 3(STAT3)/nuclear factor kappa B(NF-κB)signaling pathway,and exacerbate the inflammatory response,thus participating in the pathogenesis of ulcerative colitis(UC).Mesalazine is a commonly used drug in the clinical treatment of UC.However,further studies are needed to determine whether mesalazine regulates the ER stress of intestinal epithelial cells,downregulates the STAT3/NF-κB pathway to play a role in the treatment of UC.AIM To study the therapeutic effects of mesalazine on spontaneous colitis in interleukin-10(IL-10)-/-mice.METHODS The 24-week-old IL-10-/-mice with spontaneous colitis were divided into the model group and the 5-amino salicylic acid group.Littermates of wild-type mice of the same age group served as the control.There were eight mice in each group,four males and four females.The severity of symptoms of spontaneous colitis in IL-10-/-mice was assessed using disease activity index scores.On day 15,the mice were sacrificed.The colon length was measured,and the histopathological changes and ultrastructure of colonic epithelial cells were detected.The protein expressions of STAT3,p-STAT3,NF-κB,IκB,p-IκB,and glucoseregulated protein 78 were identified using Western blotting.The STAT3 and NF-κB mRNA expressions were identified using real-time polymerase chain reaction.The glucose-regulated protein 78 and C/EBP homologous protein expressions in colon sections were detected using immunofluorescence.RESULTS Mesalazine reduced the symptoms of spontaneous colitis in IL-10 knockout mice and the histopathological damage of colonic tissues,and alleviated the ER stress in epithelial cells of colitis mice.Western blotting and quantitative real-time polymerase chain reaction results showed that the STAT3/NF-κB pathway in the colon tissue of model mice was activated,suggesting that this pathway was involved in the pathogenesis of UC and might become a potential therapeutic target.Mesalazine could down-regulate the protein expressions of p-STAT3,NF-κB and p-IκB,and down-regulate the mRNA expression of STAT3 and NF-κB.CONCLUSION Mesalazine may play a protective role in UC by reducing ER stress by regulating the STAT3/NF-κB signaling pathway.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Hypoglycemic mechanism of Tegillarca granosa polysaccharides on type 2 diabetic mice by altering gut microbiota and regulating the PI3K-akt signaling pathwaye

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease threatening human health.We investigated the effects of Tegillarca granosa polysaccharide(TGP)and determined its potential mechanisms in a mouse model of T2DM established through a high-fat diet and streptozotocin.TGP(5.1×10^(3) Da)was composed of mannose,glucosamine,rhamnose,glucuronic acid,galactosamine,glucose,galactose,xylose,and fucose.It could significantly alleviate weight loss,reduce fasting blood glucose levels,reverse dyslipidemia,reduce liver damage from oxidative stress,and improve insulin sensitivity.RT-PCR and Western blotting indicated that TGP could activate the phosphatidylinositol-3-kinase/protein kinase B signaling pathway to regulate disorders in glucolipid metabolism and improve insulin resistance.TGP increased the abundance of Allobaculum,Akkermansia,and Bifidobacterium,restored the microbiota abundance in the intestinal tracts of mice with T2DM,and promoted short-chain fatty acid production.This study provides new insights into the antidiabetic effects of TGP and highlights its potential as a natural hypoglycemic nutraceutical.

Paeoniflorin ameliorates chronic colitis via the DR3 signaling pathway in group 3 innate lymphoid cells

Inhibiting the death receptor 3(DR3)signaling pathway in group 3 innate lymphoid cells(ILC3s)presents a promising approach for promoting mucosal repair in individuals with ulcerative colitis(UC).Paeoniflorin,a prominent component of Paeonia lactiflora Pall.,has demonstrated the ability to restore barrier function in UC mice,but the precise mechanism remains unclear.In this study,we aimed to delve into whether paeoniflorin may promote intestinal mucosal repair in chronic colitis by inhibiting DR3 signaling in ILC3s.C57BL/6 mice were subjected to random allocation into 7 distinct groups,namely the control group,the 2%dextran sodium sulfate(DSS)group,the paeoniflorin groups(25,50,and 100 mg/kg),the anti-tumor necrosis factor-like ligand 1A(anti-TL1A)antibody group,and the IgG group.We detected the expression of DR3 signaling pathway proteins and the proportion of ILC3s in the mouse colon using Western blot and flow cytometry,respectively.Meanwhile,DR3-overexpressing MNK-3 cells and 2%DSS-induced Rag1^(-/-)mice were used for verification.The results showed that paeoniflorin alleviated DSS-induced chronic colitis and repaired the intestinal mucosal barrier.Simultaneously,paeoniflorin inhibited the DR3 signaling pathway in ILC3s and regulated the content of cytokines(interleukin-17A,granulocyte-macrophage colony stimulating factor,and interleukin-22).Alternatively,paeoniflorin directly inhibited the DR3 signaling pathway in ILC3s to repair mucosal damage independently of the adaptive immune system.We additionally confirmed that paeoniflorin-conditioned medium(CM)restored the expression of tight junctions in Caco-2 cells via coculture.In conclusion,paeoniflorin ameliorates chronic colitis by enhancing the intestinal barrier in an ILC3-dependent manner,and its mechanism is associated with the inhibition of the DR3 signaling pathway.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

Oleanolic acid inhibits colon cancer cell stemness and reverses chemoresistance by suppressing JAK2/STAT3 signaling pathway

Background:Oleanolic acid(OA),a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity,was isolated from traditional Chinese medicinal herbs.Conversely,the OA that impacts colon cancer(CC)cells and its underlying mechanisms remain poorly understood.Methods:The cytotoxic effect of OA alone or OA-5-Fluorouracil(5-FU)combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide(MTT).Then,the impact of OA on CC cell lines(LoVo and HT-29)proliferation and stemness were measured using colon formation and tumorsphere formation assays.Octamer-binding transcription factor 4(Oct4),Prominin-1(CD133),Nanog,and transcription factor SOX-2(SOX2)are cell stemness-related indicators whose expression was assessed usingfluorescence qPCR assay,Western blotting,and immunohistochemistry.The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model.Results:The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU.Moreover,OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers(CD133,Nanog,SOX2,and Oct4)expression levels both in vitro and in vivo,as well as by inactivating the activator of transcription 3(STAT3 signaling)and Janus kinase 2/signal transducer(JAK2).Conclusion:Thesefindings imply that oleanolic acid,both in vitro and in vivo,suppresses the JAK2/STAT3 pathway,which in turn reverses chemoresistance and decreases colon cancer cell stemness.Therefore,by reducing the recommended amount of 5-FU,this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

Mechanism of action of cordycepin in the treatment of hepatocellular carcinoma via regulation of the Hippo signaling pathway

Hepatocellular carcinoma(HCC)is one of the common most malignant tumors.This study aimed to determine the in vitro and in vivo anticancer activity of cordycepin and elucidate its mechanism of action.The results of in vitro and in vivo studies revealed that cordycepin inhibited proliferation and migration in HepG-2 cells and inhibited the growth of HepG-2 xenograft-bearing nude mice by inducing apoptosis.Transcriptome sequencing analysis revealed a total of 403 differential genes,which revealed that cordycepin may play an anti-HCC role by regulating Hippo signaling pathway.The regulatory effects of cordycepin on the Hippo signaling pathway was further investigated using a YAP1 inhibitor.The results demonstrated that cordycepin upregulated the expression of MST1 and LAST1,and subsequently inhibited YAP1,which activated the Hippo signaling pathway.This in turn downregulated the expression of GBP3 and ETV5,and subsequently inhibited cell proliferation and migration.Additionally,YAP1 regulated the expression of Bax and Bcl-2,regulated the mitochondrial apoptotic pathway,and induced apoptosis by upregulating the expression of the caspase-3 protein.In summary,this study reveals that cordycepin exerts its anti-hepatocarcinoma effect through regulating Hippo signaling pathway,and GBP3 and ETV5 may be potential therapeutic targets for hepatocarcinoma.

Acacetin protects against cerebral ischemia-reperfusion injury via the NLRP3 signaling pathway

Acacetin(5,7-dihydroxy-4′-methoxyflavone), a potential neuroprotective agent, has an inhibitory effect on lipopolysaccharide-induced neuroinflammatory reactions. However, whether acacetin has an effect on inflammatory corpuscle 3(NLRP3) after cerebral ischemia-reperfusion injury has not been fully determined. This study used an improved suture method to establish a cerebral ischemia-reperfusion injury model in C57BL/6 mice. After ischemia with middle cerebral artery occlusion for 1 hour, reperfusion with intraperitoneal injection of 25 mg/kg of acacetin(acacetin group) or an equal volume of saline(0.1 mL/10 g, middle cerebral artery occlusion group) was used to investigate the effect of acacetin on cerebral ischemia-reperfusion injury. Infarct volume and neurological function scores were determined by 2,3,5-triphenyltetrazolium chloride staining and the Zea-Longa scoring method. Compared with the middle cerebral artery occlusion group, neurological function scores and cerebral infarction volumes were significantly reduced in the acacetin group. To understand the effect of acacetin on microglia-mediated inflammatory response after cerebral ischemia-reperfusion injury, immunohistochemistry for the microglia marker calcium adapter protein ionized calcium-binding adaptor molecule 1(Iba1) was examined in the hippocampus of ischemic brain tissue. In addition, tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in ischemic brain tissue of mice was quantified by enzyme-linked immunosorbent assay. Expression of Iba1, tumor necrosis factor-α, interleukin-1β and interleukin-6 was significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Western blot assay results showed that expression of Toll-like receptor 4, nuclear factor kappa B, NLRP3, procaspase-1, caspase-1, pro-interleukin-1β, and interleukin-1β were significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Our findings indicate that acacetin has a protective effect on cerebral ischemia-reperfusion injury, and its mechanism of action is associated with inhibition of microglia-mediated inflammation and the NLRP3 signaling pathway.

PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway

AIM: To investigate the role of pre-B-cell leukemia homeobox (PBX)3 in migration and invasion of colorectal cancer (CRC) cells.

Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury

Baicalin is a natural active ingredient isolated from Scutellariae Radix that can cross the blood-brain barrier and exhibits neuroprotective effects on multiple central nervous system diseases.However,the mechanism behind the neuroprotective effects remains unclear.In this study,rat models of spinal cord injury were established using a modified Allen's impact method and then treated with intraperitoneal injection of Baicalin.The results revealed that Baicalin greatly increased the Basso,Beattie,Bresnahan Locomotor Rating Scale score,reduced blood-spinal cord barrier permeability,decreased the expression of Bax,Caspase-3,and nuclear factorκB,increased the expression of Bcl-2,and reduced neuronal apoptosis and pathological spinal cord injury.SH-SY5 Y cell models of excitotoxicity were established by application of 10 m M glutamate for 12 hours and then treated with 40μM Baicalin for 48 hours to investigate the mechanism of action of Baicalin.The results showed that Baicalin reversed tight junction protein expression tendencies(occludin and ZO-1)and apoptosis-related protein expression(Bax,Bcl-2,Caspase-3,and nuclear factor-κB),and also led to up-regulation of PI3 K and Akt phosphorylation.These effects on Bax,Bcl-2,and Caspase-3 were blocked by pretreatment with the PI3 K inhibitor LY294002.These findings suggest that Baicalin can inhibit bloodspinal cord barrier permeability after spinal cord injury and reduce neuronal apoptosis,possibly by activating the PI3 K/Akt signaling pathway.This study was approved by Animal Ethics Committee of Xi'an Jiaotong University on March 6,2014.

Endogenous hydrogen sulfide and ERK1/2-STAT3 signaling pathway may participate in the association between homocysteine and hypertension

Background Homocysteine(Hcy)is a risk factor for hypertension,although the mechanisms are poorly understood.Methods We first explored the relationship between Hcy levels and blood pressure(BP)by analyzing the clinical data of primary hypertensive patients admitted to our hospital.Secondly,we explored a rat model to study the effect of Hcy on blood pressure and the role of H2S.An hyperhomocysteinemia(HHcy)rat model was induced to explore the effect of Hcy on blood pressure and the possible mechanism.We carried out tissue histology,extraction and examination of RNA and protein.Finally,we conducted cell experiments to determine a likely mechanism through renin-angiotensin-aldosterone system(RAAS)and extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway.Results In primary hypertensive inpatients with HHcy,blood pressure was significantly higher as compared with inpatient counterparts lacking HHcy.In the rat model,blood pressure of the Wistar rats was significantly increased with increases in serum Hcy levels and decreased after folate treatment.Angiotensin converting enzyme 1(ACE1)expression in the Wistar Hcy group was enhanced comparing to controls,but was decreased in the Wistar folate group.Angiotensin II receptor type 1(AGTR1)levels in the kidney tissue increased in the Wistar folate group.Both serum H2S and kidney cystathionineγ-lyase decreased with elevated levels of serum Hcy.In vitro,increased concentrations and treatment times for Hcy were associated with increased expression of collagen type 1 and AGTR1.This dose and time dependent response was also observed for p-STAT3 and p-ERK1/2 expression.Conclusion Endogenous H2S might mediate the process of altered blood pressure in response to changes in serum Hcy levels,in a process that is partly dependent on activated RAAS and ERK1/2-STAT3 signaling pathway.

Down-regulation of mi R-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway

AIM To investigate the potential role of micro RNA-30 a(mi R-30 a) in esophageal squamous cell carcinoma(ESCC).METHODS Expression of mi R-30 a-3 p/5 p was analyzed using microarray data and fresh ESCC tissue samples. Both in vitro and in vivo assays were used to investigate the effects of mi R-30 a-3 p/5 p on ESCC cell proliferation. Furthermore,Kyoto Encyclopedia of Genes and Genomes analysis was performed to explore underlying mechanisms involved in ESCC,and then,assays were carried out to verify the potential molecular mechanism of mi R-30 a in ESCC.RESULTS Low expression of mi R-30 a-3 p/5 p was closely associated with advanced ESCC progression and poor prognosis of patients with ESCC. Knock-down of mi R-30 a-3 p/5 p promoted ESCC cell proliferation. Increased mi R-30 a-3 p/5 p expression inhibited the Wnt signaling pathway by targeting Wnt2 and Fzd2.CONCLUSION Down-regulation of mi R-30 a-3 p/5 p promotes ESCC cell proliferation by activating the Wnt signaling pathway through inhibition of Wnt2 and Fzd2.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

BACKGROUND Atherosclerosis is a major cause of mortality worldwide and is driven by multiple risk factors,including diabetes,which results in an increased atherosclerotic burden,but the precise mechanisms for the occurrence and development of diabetic atheroscerosis have not been fully elucidated.AIM To summarize the potential role of retinol binding protein 4(RBP4) in the pathogenesis of diabetic atheroscerosis,particularly in relation to the RBP4-Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.METHODS Male Wistar rats were randomly divided into three groups,including a control group(NC group),diabetic rat group(DM group),and diabetic atherosclerotic rat group(DA group).The contents of total cholesterol(TC), high-density lipoprotein cholesterol(HDL-c), triglycerides(TG), low-density lipoprotein cholesterol(LDLc), fasting insulin(FINS),fasting plasma glucose,and hemoglobin A1 c(HbA1 c)were measured.Moreover,the adipose and serum levels of RBP4,along with the expression levels of JAK2, phosphorylated JAK2(p-JAK2), STAT3,phosphorylated STAT3(p-STAT3), B-cell lymphoma-2(Bcl-2), and Cyclin D1 in aortic tissues were also measured.Besides,homeostasis model assessment of insulin resistance(HOMA-IR) and atherogenic indexes(AI) were calculated.RESULTS Compared with the NC and DM groups,the levels LDL-c,TG,TC,FINS,HOMAIR,RBP4,and AI were upregulated,whereas that of HDL-c was downregulated in the DA group(P <0.05);the mRNA levels of JAK2,STAT3,Cyclin D1,and Bcl-2 in the DA group were significantly increased compared with the NC group and the DM group;P-JAK2,p-JAK2/JAK2 ratio,p-STAT3,p-STAT3/STAT3 ratio,Cyclin D1,and Bcl-2 at protein levels were significantly upregulated in the DA group compared with the NC group and DM group.In addition,as shown by Pearson analysis,serum RBP4 had a positive correlation with TG,TC,LDL-c,FINS,HbA1 C,p-JAK2,p-STAT3,Bcl-2,Cyclin D1,AI,and HOMA-IR but a negative correlation with HDL-c.In addition,multivariable logistic regression analysis showed that serum RBP4,p-JAK2,p-STAT3,and LDL-c were predictors of the presence of diabetic atherosclerosis.CONCLUSION RBP4 could be involved in the initiation or progression of diabetic atherosclerosis by regulating the JAK2/STAT3 signaling pathway.

shRNA-interfering LSD1 inhibits proliferation and invasion of gastric cancer cells via VEGF-C/PI3K/AKT signaling pathway

BACKGROUND Histone Lysine Specific Demethylase 1(LSD1)is the first histone demethylase to be discovered,which regulates various biological functions by making lysine of histone H3K4,H3K9 and non-histone substrates demethylated.Abnormal regulation of LSD1 is closely related to the occurrence and development of gastric cancer.The change of LSD1 expression level plays an important role in the proliferation and metastasis of gastric cancer cells.The study of its function and mechanism may provide a theoretical basis for early diagnosis and targeted therapy of gastric cancer.AIM To investigate the effect of downregulation of lysine-specific demethylase 1(LSD1)expression on proliferation and invasion of gastric cancer cells and the possible regulatory mechanisms of the VEGF-C/PI3K/AKT signaling pathway.METHODS The LSD1-specific short hairpin RNA(shRNA)interference plasmid was transiently transfected,and expression of LSD1 was downregulated.The cell proliferation ability of LSD1 was observed by CCK-8 assay after downregulating expression of LSD1.Transwell invasion assay was used to observe the change of cell invasion ability after downregulating expression of LSD1.Expression of phosphorylated phosphoinositide 3-kinase(p-PI3K),PI3K,p-AKT,AKT,vascular endothelial growth factor receptor(VEGFR)-3,matrix metalloproteinase(MMP)-2 and MMP-9 in each group was detected by Western blotting.RESULTS The cell proliferation ability of transiently transfected LSD1-shRNA interference plasmid group was significantly lower than that of the control group(P<0.05).Transwell invasion assay showed that the number of cells across the membrane of the LSD1-shRNA transfection group(238.451±5.216)was significantly lower than that of the control group(49.268±6.984)(P<0.01).Western blotting showed that expression level of VEGF-C,p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2 and MMP-9 in the LSD1-shRNA group was significantly lower than that in the control group(P<0.05).CONCLUSION Downregulation of LSD1 expression inhibits metastatic potential of gastric cancer cells,and VEGF-C-mediated activation of PI3K/AKT signaling pathway,which may be an important mechanism for inhibiting lymph node metastasis in gastric cancer cells.

MiR-19a-3p regulates the Forkhead box F2-mediated Wnt/β-catenin signaling pathway and affects the biological functions of colorectal cancer cells

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.AIM To explore the expression of microRNA miR-19a-3p and Forkhead box F2(FOXF2)in patients with CRC and the relevant mechanisms.METHODS Sixty-two CRC patients admitted to the hospital were enrolled into the study group,and sixty healthy people from the same period were assigned to the control group.Elbow venous blood was sampled from the patients and healthy individuals,and blood serum was saved for later analysis.MiR-19a-3p mimics,miR-19a-3p inhibitor,miR-negative control,small interfering-FOXF2,and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells.Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells,and western blot(WB)analysis was conducted to evaluate the levels of FOXF2,glycogen synthase kinase 3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,p-β-catenin,α-catenin,Ncadherin,E-cadherin,and vimentin.The MTT,Transwell,and wound healing assays were applied to analyze cell proliferation,invasion,and migration,respectively,and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.RESULTS The patients showed high serum levels of miR-19a-3p and low levels of FOXF2,and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8.MiR-19a-3p and FOXF2 were related to sex,tumor size,age,tumor-nodemetastasis staging,lymph node metastasis,and differentiation of CRC patients.Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelialmesenchymal transition,invasion,migration,and proliferation of cells.WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β,β-catenin,N-cadherin,and vimentin;and increased the levels of GSK-3β,p-β-catenin,α-catenin,and Ecadherin.The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2.In addition,a rescue experiment revealed that there were no differences in cell proliferation,invasion,and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.CONCLUSION Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway,thereby affecting the epithelial-mesenchymal transition,proliferation,invasion,and migration of cells.Thus,miR-19a-3p is likely to be a therapeutic target in CRC.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

Scoparone inhibits pancreatic cancer through PI3K/Akt signaling pathway

BACKGROUND Pancreatic cancer is a highly malignant tumor of the gastrointestinal system whose emerging resistance to chemotherapy has necessitated the development of novel antitumor treatments.Scoparone,a traditional Chinese medicine monomer with a wide range of pharmacological properties,has attracted considerable attention for its antitumor activity.AIM To explore the potential antitumor effect of scoparone on pancreatic cancer and the possible molecular mechanism of action.METHODS The target genes of scoparone were determined using both the bioinformatics and multiplatform analyses.The effect of scoparone on pancreatic cancer cell proliferation,migration,invasion,cell cycle,and apoptosis was detected in vitro.The expression of hub genes was tested using quantitative reverse transcription polymerase chain reaction(qRT-PCR),and the molecular mechanism was analyzed using Western blot.The in vivo effect of scoparone on pancreatic cancer cell proliferation was detected using a xenograft tumor model in nude mice as well as immunohistochemistry.RESULTS The hub genes involved in the suppression of pancreatic cancer by scoparone were obtained by network bioinformatics analyses using publicly available databases and platforms,including SwissTargetPrediction,STITCH,GeneCards,CTD,STRING,WebGestalt,Cytoscape,and Gepia;AKT1 was confirmed using qRT-PCR to be the hub gene.Cell Counting Kit-8 assay revealed that the viability of Capan-2 and SW1990 cells was significantly reduced by scoparone treatment exhibiting IC50 values of 225.2μmol/L and 209.1μmol/L,respectively.Wound healing and transwell assays showed that scoparone inhibited the migration and invasion of pancreatic cancer cells.Additionally,flow cytometry confirmed that scoparone caused cell cycle arrest and induced apoptosis.Scoparone also increased the expression levels of Bax and cleaved caspase-3,decreased the levels of MMP9 and Bcl-2,and suppressed the phosphorylation of Akt without affecting total PI3K and Akt.Moreover,compared with the control group,xenograft tumors,in the 200μmol/L scoparone treatment group,were smaller in volume and lighter in weight,and the percentages of Ki65-and PCNA-positive cells were decreased.CONCLUSION Our findings indicate that scoparone inhibits pancreatic cancer cell proliferation in vitro and in vivo,inhibits migration and invasion,and induces cycle arrest and apoptosis in vitro through the PI3K/Akt signaling pathway.