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.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Human neural stem cell-derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

Human neural stem cell-derived extracellular vesicles exhibit analogous functions to their parental cells,and can thus be used as substitutes for stem cells in stem cell therapy,thereby mitigating the risks of stem cell therapy and advancing the frontiers of stem cell-derived treatments.This lays a foundation for the development of potentially potent new treatment modalities for ischemic stroke.However,the precise mechanisms underlying the efficacy and safety of human neural stem cell-derived extracellular vesicles remain unclear,presenting challenges for clinical translation.To promote the translation of therapy based on human neural stem cell-derived extracellular vesicles from the bench to the bedside,we conducted a comprehensive preclinical study to evaluate the efficacy and safety of human neural stem cell-derived extracellular vesicles in the treatment of ischemic stroke.We found that administration of human neural stem cell-derived extracellular vesicles to an ischemic stroke rat model reduced the volume of cerebral infarction and promoted functional recovery by alleviating neuronal apoptosis.The human neural stem cell-derived extracellular vesicles reduced neuronal apoptosis by enhancing phosphorylation of phosphoinositide 3-kinase,mammalian target of rapamycin,and protein kinase B,and these effects were reversed by treatment with a phosphoinositide 3-kinase inhibitor.These findings suggest that human neural stem cell-derived extracellular vesicles play a neuroprotective role in ischemic stroke through activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.Finally,we showed that human neural stem cell-derived extracellular vesicles have a good in vivo safety profile.Therefore,human neural stem cell-derived extracellular vesicles are a promising potential agent for the treatment of ischemic stroke.

Xuebijing improves intestinal microcirculation dysfunction in septic rats by regulating the VEGF-A/PI3K/Akt signaling pathway

BACKGROUND:This study aims to explore whether Xuebijing(XBJ) can improve intestinal microcirculation dysfunction in sepsis and its mechanism.METHODS:A rat model of sepsis was established by cecal ligation and puncture(CLP).A total of 30 male SD rats were divided into four groups:sham group,CLP group,XBJ + axitinib group,and XBJ group.XBJ was intraperitoneally injected 2 h before CLP.Hemodynamic data(blood pressure and heart rate) were recorded.The intestinal microcirculation data of the rats were analyzed via microcirculation imaging.Enzyme-linked immunosorbent assay(ELISA) kits were used to detect the serum levels of interleukin-6(IL-6),C-reactive protein(CRP),and tumor necrosis factor-α(TNF-α) in the rats.Histological analysis and transmission electron microscopy were used to analyze the injury of small intestinal microvascular endothelial cells and small intestinal mucosa in rats.The expression of vascular endothelial growth factor A(VEGF-A),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt) in the small intestine was analyzed via Western blotting.RESULTS:XBJ improved intestinal microcirculation dysfunction in septic rats,alleviated the injury of small intestinal microvascular endothelial cells and small intestinal mucosa,and reduced the systemic inflammatory response.Moreover,XBJ upregulated the expression of VEGF-A,p-PI3K/total PI3K,and p-Akt/total Akt in the rat small intestine.CONCLUSION:XBJ may improve intestinal microcirculation dysfunction in septic rats possibly through the VEGF-A/PI3K/Akt signaling pathway.

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions

Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.

Roles of the PI3K/Akt pathway in Epstein-Barr virusinduced cancers and therapeutic implications

Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus(EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins(LMP) 1 and LMP2 A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3 K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3 K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

Banxia xiexin decoction prevents the development of gastric cancer

BACKGROUND In China banxia xiexin decoction(BXD)has been used in treating gastric cancer(GC)for thousands of years and BXD has a good role in reversing GC histopathology,but its chemical composition and action mechanism are still unknown.AIM To investigate the mechanism of action of BXD against GC based on transcriptomics,network pharmacology,in vivo and in vitro experiments.METHODS The transplanted tumor model was prepared,and the nude mouse were pathologically examined after administration,and hematoxylin-eosin staining was performed.The active ingredients of BXD were quality controlled and identified using ultra-performance liquid chromatography tandem quadrupole electrostatic field orbitrap mass spectrometry(UPLC-Q-Orbitrap MS/MS),and traditional Chinese medicines systems pharmacology platform,drug bank and the Swiss target prediction platform to predict the relevant targets,the differentially expressed genes(DEGs)of GC were screened by RNA-seq sequencing,and the overlapping targets were analyzed to obtain the key targets and pathways.Cell Counting Kit-8,apoptosis assay,cell migration and Realtime fluorescence quantitative polymerase chain reaction were used for in vitro experiments.RESULTS All dosing groups inhibited the growth of transplanted tumors in laboratory-bred strain nude,with the capecitabine group and the BXD medium-dose group being the best.A total of 29 compounds and 859 potential targets in BXD were identified by UPLC-Q-Orbitrap MS/MS and network pharmacology,RNA-seq sequencing found 4767 GC DEGs,which were combined with network pharmacology and analyzed 246 potential therapeutic targets were obtained and pathway results showed that BXD may against GC through the Phosphoinositide 3-kinase(PI3K)/protein kinase B(AKt)signaling pathway.In vitro cellular experiments confirmed that BXDcontaining serum and LY294002 could inhibit the proliferation of GC cells,promote apoptosis,and inhibit the migration of GC cells by decreasing the expression of EGFR,PIK3CA,IL6,BCL2 and AKT1 in the PI3K-Akt pathway in MGC-803 expression.CONCLUSION BXD has the effect of inhibiting tumor growth rate and delaying the development of GC.Its mechanism of action may be related to the regulation of PI3K-Akt signaling pathway.

PI_3K/Akt信号通路在神经系统疾病中的研究进展

磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路具有调节细胞增殖、分化、代谢等多种作用。现就近年来有关PI3K/Akt信号通路在神经系统疾病中的研究进展作一综述,客观评价PI3K/Akt信号通路在脑血管疾病、神经变性疾病、脱髓鞘疾病、运动障碍性疾病、癫痫和肌肉疾病的研究状况,为探索神经系统疾病新的发病机制、寻找新的治疗方法和诊断指标提供理论依据。

Tongxinluo Activates PI3K/AKT Signaling Pathway to Inhibit Endothelial Mesenchymal Transition and Attenuate Myocardial Fibrosis after Ischemia-Reperfusion in Mice

Objective To investigate the potential role of Tongxinluo(TXL)in attenuating myocardial fibrosis after myocardial ischemia-reperfusion injury(MIRI)in mice.Methods A MIRI mouse model was established by left anterior descending coronary artery ligation for 45 min.According to a random number table,66 mice were randomly divided into 6 groups(n=11 per group):the sham group,the model group,the LY-294002 group,the TXL group,the TXL+LY-294002 group and the benazepril(BNPL)group.The day after modeling,TXL and BNPL were administered by gavage.Intraperitoneal injection of LY-294002 was performed twice a week for 4 consecutive weeks.Echocardiography was used to measure cardiac function in mice.Masson staining was used to evaluate the degree of myocardial fibrosis in mice.Qualitative and quantitative analysis of endothelial mesenchymal transition(EndMT)after MIRI was performed by immunohistochemistry,immunofluorescence staining and flow cytometry,respectively.The protein expressions of platelet endothelial cell adhesion molecule-1(CD31),α-smoth muscle actin(α-SMA),phosphatidylinositol-3-kinase(PI3K)and phospho protein kinase B(p-AKT)were assessed using Western blot.Results TXL improved cardiac function in MIRI mice,reduced the degree of myocardial fibrosis,increased the expression of CD31 and inhibited the expression ofα-SMA,thus inhibited the occurrence of EndMT(P<0.05 or P<0.01).TXL significantly increased the protein expressions of PI3K and p-AKT(P<0.05 or P<0.01).There was no significant difference between TXL and BNPL group(P>0.05).In addition,the use of the PI3K/AKT pathway-specific inhibitor LY-294002 to block this pathway and combination with TXL intervention,eliminated the protective effect of TXL,further supporting the protective effect of TXL.Conclusion TXL activated the PI3K/AKT signaling pathway to inhibit EndMT and attenuated myocardial fibrosis after MIRI in mice.

Scorpiones,Scolopendra and Gekko Inhibit Lung Cancer Growth and Metastasis by Ameliorating Hypoxic Tumor Microenvironment via PI3K/AKT/mTOR/HIF-1αSignaling Pathway

Objective:To investigate whether Buthus martensii karsch(Scorpiones),Scolopendra subspinipes mutilans L.Koch(Scolopendra)and Gekko gecko Linnaeus(Gekko)could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α(PI3K/AKT/mTOR/HIF-1α)signaling pathway.Methods:Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models,with rapamycin and cyclophosphamide as positive controls.Carboxy methyl cellulose solutions of Scorpiones,Scolopendra and Gekko were administered intragastrically as 0.33,0.33,and 0.83 g/kg,respectively once daily for 21 days.Fluorescent expression were detected every 7 days after inoculation,and tumor growth curves were plotted.Immunohistochemistry was performed to determine CD31 and HIF-1αexpressions in tumor tissue and microvessel density(MVD)was analyzed.Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1αsignaling pathway-related proteins.Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor(bFGF),transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)in mice.Results:Scorpiones,Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α(all P<0.01).Moreover,Scorpiones,Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase(p70S6K)(P<0.05 or P<0.01).In addition,they also decreased the expression of CD31,MVD,bFGF,TGF-β1 and VEGF compared with the model group(P<0.05 or P<0.01).Conclusion:Scorpiones,Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1αsignaling pathway.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

Elucidation of the hepatoprotective effect and mechanism of Melastoma dodecandrum Lour. based on network pharmacology and experimental validation

Objective:To systematically explore the effect and mechanism of melastomatis dodecandri herba(Melastoma dodecandrum Lour.)in the treatment of hepatitis based on network pharmacology.Method:We evaluated the hepatoprotective effects of M.dodecandrum in concanavalin A(Con A)-induced hepatitis in mice by assessing survival rate,histological analysis,serum transaminases,and related cytokines.Then the mechanism of action was predicted by a network pharmacology-based strategy.Based on the results,we measured the hepatic expression of related genes at mRNA level and proteins related to the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)and nuclear factorkappa B(NF-кB)pathways.Results:Our study results clearly demonstrated that M.dodecandrum pretreatment significantly alleviated liver injury.This was demonstrated by an increase in survival rate,decreased severity of liver damage,and reduced serum transaminase levels compared with those in the Con A group.Moreover,M.dodecandrum significantly reduced the serum levels of tumor necrosis factor-a,interleukin-6,and interferon-g and increased the liver levels of superoxide dismutase,which indicated that M.dodecandrum exhibits anti-inflammatory and antioxidant activities.On the basis of network pharmacology,50 nodes were selected as major hubs based on their topological importance.Pathway enrichment analyses indicated that the putative targets of M.dodecandrum mostly participate in various pathways associated with the anti-inflammation response,which implies the underlying mechanism by which M.dodecandrum acts on hepatitis.Real-time fluorescent quantitative PCR analysis showed that M.dodecandrum downregulates the mRNA expression of interleukin-6,Toll-like receptor 7,interleukin-1 receptor-associated kinase-4,NF-кB and tumor necrosis factor-a in liver tissues.Western blotting showed that M.dodecandrum pretreatment protected against inflammation through activating the PI3K-Akt pathway by upregulating phosphorylated Akt(p-Akt)expression and suppressing NF-кB activation by inhibiting the phosphorylation of IKK,IkBa,and p65.Conclusion:The present work demonstrated the hepatoprotective effects of M.dodecandrum by regulating the PI3K/Akt and NF-кB pathways in Con A-induced mice,which provide insights into the treatment of hepatitis using M.dodecandrum.

Liposomalα-cyperone targeting bone resorption surfaces suppresses osteoclast differentiation and osteoporosis progression via the PI3K/Akt axis

Osteoporosis is a metabolic dysregulation of bone that occurs mainly in postmenopausal women,and the hyperfunction of osteoclasts is the primary contributor to postmenopausal osteoporosis.However,the development of effective therapeutic drugs and precise delivery systems remains a challenge in the field of anti-absorption therapy.Here,we reported theα-cyperone(α-CYP)for anti-osteoporosis and developed a liposome-based nano-drug delivery system ofα-CYP,that specifically targets the bone resorption interface.Firstly,we found that theα-CYP,one of the major sesquiterpenes of Cyperus rotundus L.,attenuated the progression of osteoporosis in ovariectomized(OVX)mice and down-regulated the expression of phosphorylated proteins of phosphoinositide 3-kinase(PI3K)and protein kinase B(Akt),causing down-regulation of osteoclast-related genes/proteins and curbing osteoclast differentiation.Furthermore,α-CYP reversed the activation of osteoclastic differentiation and enhanced osteoporosis-related proteins expression caused by PI3K/Akt agonist(YS-49).More importantly,we adopted the osteoclastic resorption surface targeting peptide Asp8 and constructed the liposome(lipαC@Asp8)to deliverα-CYP to osteoclasts and confirmed its anti-osteoporosis effect and enhanced osteoclast inhibition by blocking PI3K/Akt axis.In conclusion,this study demonstrated thatα-CYP inhibits osteoclast differentiation and osteoporosis development by silencing PI3K/Akt pathway,and the liposome targeting delivery systems loaded withα-CYP might provide a novel and effective strategy to treat osteoporosis.

Treating non-small cell lung cancer by targeting the PI3K signaling pathway

The phosphosphatidylinositol-3-kinase(PI3K)signaling pathway is one of the most important intracellular signal transduction pathways affecting cell functions,such as apoptosis,translation,metabolism,and angiogenesis.Lung cancer is a malignant tumor with the highest morbidity and mortality rates in the world.It can be divided into two groups,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for>85%of all lung cancers.There are currently many clinical treatment options for NSCLC;however,traditional methods such as surgery,chemotherapy,and radiotherapy have not been able to provide patients with good survival benefits.The emergence of molecular target therapy has improved the survival and prognosis of patients with NSCLC.In recent years,there have been an increasing number of studies on NSCLC and PI3K signaling pathways.Inhibitors of various parts of the PI3K pathway have appeared in various phases of clinical trials with NSCLC as an indication.This article focuses on the role of the PI3K signaling pathway in the occurrence and development of NSCLC and summarizes the current clinical research progress and possible development strategies.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair

Objective:To investigate the effects of Danmu Extract Syrup(DMS) on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice and explore the mechanism.Methods:Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table(n=12),including control(normal saline),LPS(5 mg/kg),LPS+DMS 2.5 mL/kg,LPS+DMS 5 mL/kg,LPS+DMS 10 mL/kg,and LPS+Dexamethasone(DXM,5 mg/kg) groups.After pretreatment with DMS and DXM,the ALI mice model was induced by LPS,and the bronchoalveolar lavage fluid(BALF) were collected to determine protein concentration,cell counts and inflammatory cytokines.The lung tissues of mice were stained with hematoxylin-eosin,and the wet/dry weight ratio(W/D) of lung tissue was calculated.The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1βin BALF of mice were detected by enzyme linked immunosorbent assay.The expression levels of Claudin-5,vascular endothelial(VE)-cadherin,vascular endothelial growth factor(VEGF),phospho-protein kinase B(p-Akt) and Akt were detected by Western blot analysis.Results:DMS pre-treatment significantly ameliorated lung histopathological changes.Compared with the LPS group,the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment(P<0.05 or P<0.01).The number of cells in BALF and myeloperoxidase(MPO) activity decreased significantly after DMS pretreatment(P<0.05 or P<0.01).DMS pre-treatment decreased the levels of TNF-α,IL-6 and IL-1β(P<0.01).Meanwhile,DMS activated the phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) pathway and reversed the expressions of Claudin-5,VE-cadherin and VEGF(P<0.01).Conclusions:DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier.It might be a potential therapeutic drug for LPS-induced lung injury.

Role and mechanism of matrine alone and combined with acitretin for HaCaT cells and psoriasis-like murine models

Background: Acitretin and matrine have been used in the treatment of psoriasis in China. This study was designed to investigate the role and related mechanisms of matrine alone and in combination with acitretin in the treatment of psoriasis in vitro and in vivo. Methods: HaCaT cells were treated with matrine at different concentrations of 0 (blank control), 0.2, 0.4, 0.8, and 1.6 mg/mL for 24, 48, 72 h, respectively. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay was used to assess the growth and proliferation of HaCaT cells. Cell cycle and apoptosis were detected by flow cytometry. Expression of protein was detected by Western blotting. Autophagy was observed by transmission electron microscopy. Then HaCaT cells were assigned to normal saline (NS) control group, matrine (0.4 mg/mL) group, acitretin (10 μmol/L) group, and matrine plus acitretin group, and the above methods were repeated. In animal experiments, the cumulative score (erythema, scaling, thickening) as a measure of the severity of inflammation was used to measure the skin performance of mice after treated with matrine 50 mg/kg, acitretin 4.5 mg/kg or combination of the two drugs on the psoriasis-like mouse models, respectively. Pathological findings of the lesions were observed, and the protein expressions in the lesions were detected by immunohistochemistry. Results: Cell proliferation inhibition was seen in HaCaT cells with treatment of matrine in a dose- and time-dependent manner (P < 0.01, respectively). Cell cycle G0/G1 phase arrest was observed in a dose-dependent way (P < 0.01). The expression of p21 (P < 0.05), LC3II/I (P < 0.01), and Beclin 1 (P < 0.01) increased and the expression of cyclin D1 (P < 0.05) decreased with increasing doses of matrine. Compared with the blank control, more autophagosomes were seen in HaCaT cells treated with matrine at 0.4 mg/mL by transmission electron microscopy (2.667 ± 1.202 vs. 21.33 ± 1.453, t = 9.899, P < 0.01). Cell proliferation inhibition and degree of the G0/G1 phase arrest was significantly higher in matrine plus acitretin group than those in matrine, acitretin, or the NS control group (P < 0.01, respectively). Compared with matrine or acitretin group, the expression of p21 (P < 0.05, P < 0.05) and LC3II/I (P < 0.01, P < 0.05) in matrine plus acitretin group increased significantly and the expression of cyclin D1 (P < 0.01, P < 0.05) and p62 (P < 0.05, P < 0.05) was reduced significantly. Compared with matrine or acitretin, matrine plus acitretin significantly down-regulated the phosphorylation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway (P < 0.05) and its downstream p-p70S6K (P < 0.05). In addition, the cumulative score of mice in the matrine plus acitretin group was significantly better than that in the matrine or acitretin group (1.480 ± 0.230 vs. 2.370 ± 0.241, P < 0.01;1.480 ± 0.230 vs. 2.888 ± 0.341, P < 0.01). The expression of LC3 protein in the matrine plus acitretin group was also higher than that in the matrine, acitretin, or the NS control group (P < 0.05, respectively). Conclusions: Matrine has therapeutic potentials for psoriasis. Matrine and acitretin show synergistic effect via cell cycle arrest and autophagy induction by PI3K/Akt/mTOR pathway.