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This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 μg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 μg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 μg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.
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AIM:To observe how total flavonoids of Pterocarya hupehensis Skan(PHSTF)affects the migra-tion and invasion of human rheumatoid fibroblast-like synoviocyte line MH7A.METHODS:The MH7A cells were divided into control group(without any treatment),low-,medium-and high-dose(6.25,12.5 and 25 mg/L,respectively)PHSTF groups,phosphatidylinositol 3-kinase(PI3K)inhibitor 740Y-P(10 μmol/L)group,and 740Y-P(10 μmol/L)+high-dose(25 mg/L)PHSTF group.The viability of the MH7A cells was determined by CCK-8 assay,while the migration and inva-sion were assessed by scratch and Transwell assays.The protein levels of matrix metalloproteinase-2(MMP-2),MMP-9,PI3K,p-PI3K,AKT and p-AKT were detected by Western blot.RESULTS:Compared with control group,the cell via-bility in PHSTF treatment groups was reduced(P<0.05),the cell wound healing area was significantly decreased(P<0.01),migratory and invasive cells in the lower chamber were significantly reduced(P<0.01),and the protein expres-sion of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT were decreased(P<0.01).Compared with high-dose PHSTF group,the addition of PI3K/AKT pathway agonist 740Y-P significantly increased the migration and invasion ability of MH7A cells(P<0.01)and elevated the protein expression of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT(P<0.01)under the treatment with PHSTF.CONCLUSION:Total flavonoids of Pterocarya hupehensis Skan could inhibit the migration and invasion of MH7A cells by regulating the PI3K/AKT signaling pathway.
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Objective To explore whether ferulic acid can inhibit the progression of T-cell acute lymphoblastic leukemia in vivo and in vitro by regulating PTEN/PI3K/AKT signaling pathway.Methods The T-cell acute lymphoblastic leukemia Jurkat cells were divided into the control group,the ferulic acid treatment group and the LY294002 treatment group for in vitro experiment.The cells in the control group were given normal culture;cells in the ferulic acid treatment group were given different concentrations(1.25,2.5,5,10,20,40,80,160 μmol/L)of ferulic acid,respectively,and the cell proliferation was detected by CCK-8 method,to screen the experimental concentration;cells in the LY294002 treatment group were given 50 μmol/L PI3K/AKT inhibitor LY294002.The cells proliferation,apoptosis and invasion were detected by clone formation assay,flow cytometry and Transwell assay.The relative expression levels of nuclear protein Ki67,proliferating cell nuclear antigen(PCNA),cleaved caspase-3,cleaved caspase-9,E-cadherin,N-cadherin,Vimentin,PTEN,p-PI3K,PI3K,p-AKT and AKT proteins were detected by Western blot.The nude mice models of transplanted tumors were constructed by 30 male BALB/c nude mice,and they were averagely divided into the normal group and the ferulic acid treatment group for in vivo experiment.The normal group was given normal saline by gavage,while the ferulic acid treatment group was given 75 mg/kg ferulic acid by gavage after inoculating Jurkat cells.The weight and volume changes of transplanted tumors were compared,and the levels of Ki67,cleaved caspase-3/caspase-3,E-cadherin,N-cadherin,PTEN,p-PI3K,PI3K,p-AKT and AKT in tumor tissues were detected.Results In vitro experiment,compared with the control group,the clone formation rate of cells,number of invasion cells,Ki67,PCNA,N-cadherin,Vimentin,p-PI3K/PI3K and p-AKT/AKT in the 5,10,20 μmol/L ferulic acid treatment group and the LY294002 treatment group were significantly decreased(P<0.05),while the apoptosis rate,cleaved caspase-3/caspase-3,cleaved caspase-9/caspase-9,E-cadherin and PTEN were significantly increased(P<0.05).In vivo experiment,compared with the normal group,the weight and volume of tumors were reduced in the ferulic acid treatment group,Ki67,N-cadherin,p-PI3K/PI3K and p-AKT/AKT in tumor tissues were significantly decreased,cleaved caspase-3/caspase-3,E-cadherin and PTEN were significantly increased,with statistically significant differences(P<0.05).Conclusion Ferulic acid can inhibit the proliferation and invasion of T-cell acute lymphoblastic leukemia Jurkat cells in vivo and in vitro,and induce apoptosis,its mechanism may be related to the regulation of PTEN/PI3K/AKT signaling pathway.
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Objective To investigate the protective effect and mechanism of propofol on the blood-brain barrier in rats with cerebral ischemia.Methods A total of 48 10-week-old male SD rats were randomly divided into the sham group,the cerebral ischemia group,the propofol group and the propofol+LY294002 group.Twenty-four hours before the induction of the model,the rats in the propofol+LY294002 group were intracerebroventricularly injected with PI3K inhibitor LY294002(0.3 mg·kg-1),and the rats in the other groups were administrated with saline(10 μL).Rats in the cerebral ischemia group,the propofol group and the propofol+LY294002 group established cerebral ischemia models by carotid artery occlusion.Rats in the sham group only isolated the common carotid artery and ligated the external carotid artery without other treatment.During the modeling period,the rats in the propofol group and the propofol+LY294002 group were given propofol(10 mg·kg-1)via the tail vein,and the sham group and the propofol group were treated with saline.After 24 hours,the neurological function of rats was evaluated by Zea Longa method;the area of cerebral infarction was detected by TTC staining;the degree of cerebral edema was detected by the dry-wet weight method.EB tracer method was used to evaluate the integrity of the blood-brain barrier;ELISA was used to detect inflammatory cytokines in cerebrospinal fluid;Western blot was used to detect the expression of PI3K/AKT signaling pathway proteins and blood-brain barrier tight junction proteins Claudin-5 and Occludin.Results Cerebral ischemia led to the increase of neurological function scores and local infarction of brain tissues in rats.Compared with the sham group,the EB content in the brain tissue of rats in the cerebral ischemia group increased,the degree of brain edema increased,and the content of inflammatory cytokines in the cerebrospinal fluid increased.And the use of propofol could significantly decrease the neurological function scores,reduce the area of cerebral infarction,inhibit EB penetrating blood-brain barrier,reduce the degree of brain edema,reduce the release of inflammatory cytokines,and up-regulate the expression of PI3K/AKT signaling pathway proteins and tight junction proteins Claudin-5 and Occludin.LY294002 significantly reversed the above effects of propofol.Conclusion Propofol can maintain the expression of tight junction proteins Claudin-5 and Occludin through the PI3K/AKT signaling pathway,protect the structural and functional integrity of blood-brain barrier,reduce the degree of brain edema,prevent other inflammatory cytokines into the brain tissue,reduce cerebral infarction,and alleviate the neurological functional damage caused by cerebral ischemia.
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Objective To explore the molecular mechanism of Xiaojin Pills in the treatment of breast cancer using an integrated network pharmacology and experimental verification.Methods The chemical components and potential targets of Xiaojin Pills were obtained from TCMSP,TCM-ID,ETCM and SwissTargetPrediction databases.Breast cancer related targets were collected from GeneCards,OMIM and KEGG databases.The overlapped targets were imported into STRING database to analysis a protein-protein interaction(PPI).The key targets of PPI networks were screened based on node topology parameter values through Cytoscape 3.8.0.DAVID database was used to analyze the GO and KEGG pathway enrichment to build drug-chemical components-key targets-signaling pathway network.The breast cancer cell lines MDA-MB-231 and SK-BR-3 were used to study the effects of Xiaojin Pills extract on cell apoptosis,migration and invasion,and to verify the key pathway obtained by enrichment analysis.Results Totally 181 chemical components in Xiaojin Pills were obtained,including quercetin,myricetin,pinocembrin and β-sitosterol.615 potential targets were identified for the anti-breast cancer effects of Xiaojin Pills.After overlapping,170 key targets against breast cancer were identified based on the topological analysis,which included SRC,ERK1/2,AKT1,EGFR,etc.KEGG analysis enriched pathways including pathways in cancer,MAPK signaling pathway,endocrine resistance,PI3K-AKT signaling pathway,EGFR tyrosine kinase inhibitor resistance,apoptosis,and HIF-1 signaling pathway,which may play important roles in the therapeutic effects of Xiaojin Pills against breast cancer.GO enrichment was involved in protein phosphorylation,inflammatory response,negative regulation of apoptosis,and positive regulation of ERK1 and ERK2 cascades.Cell experiments showed that Xiaojin Pills further induced mitochondria-dependent apoptosis by inhibiting the activation of MAPK and PI3K-AKT pathways.At the same time,the expressions of ZO-1 and β-catenin increased,and the epithelial-mesenchymal transformation process was reversed to inhibit the metastasis of breast cancer cells.Conclusion The key targets and signaling pathways of Xiaojin Pills in the treatment of breast cancer are studied through network pharmacology combined with in vitro experiments,which provided a basis for further study of its pharmacodynamic material basis,mechanism of action and clinical application.
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Ulcerative colitis(UC)is a common disease of the digestive system.Phosphatidylinositol-3-kinase(PI3K)/synuclein/threonine kinase(AKT)is closely related to cell survival,apoptosis,inflammation and other biological processes,and the expression levels of PI3K and AKT significantly increase during the course of UC,with accelerated apoptosis,improved inflammation,and damaged intestinal mucosal barrier function.In recent years,a large number of basic and clinical trials have been conducted on PI3K/AKT signaling pathway in TCM,and the results indicate that PI3K/AKT signaling pathway is expected to be an important potential target for UC treatment.This article analyzed the mechanism of the regulation of PI3K/AKT signaling pathway in TCM from monomer,extract,compound and acupuncture,and suggested that the regulation of this signaling pathway is of great significance for the prevention and treatment of UC,and provide reference for drug development.
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Objective To explore the potential mechanism underlying the treatment of pediatric asthma using Xiaoer Zhixiao Pingchuan Granules through network pharmacology analysis and animal experimental validation.Methods Active components and their associated targets in Xiaoer Zhixiao Pingchuan Granules were identified through screening and retrieval of TCMSP,BATMAN-TCM,and UniProt databases.Disease-related targets for pediatric asthma were selected from GeneCards,DisGeNET,and OMIM databases.The target protein-protein interaction(PPI)relationship between the intersecting targets of the two was obtained through the STRING database,and import it into Cytoscape 3.8.0 software to construct a PPI network.GO and KEGG enrichment analyses were conducted using the Metascape platform to identify potential pathways.An asthmatic mouse model was induced by ovalbumin,and different concentrations of Xiaoer Zhixiao Pingchuan Granules were administered as interventions.Histopathological changes were evaluated using HE staining and PAS staining,and the network pharmacology findings were validated through Western blot analysis.Results A total of 154 active ingredients targeting 283 pediatric asthma-related genes were identified in Xiaoer Zhixiao Pingchuan Granules.KEGG enrichment analyses revealed significant enrichment of signaling pathways such as the PI3K-Akt signaling pathway,TNF signaling pathway,and MAPK signaling pathway among intersection targets.Thirteen key targets were identified through topological analysis of ingredients-targets-pathways network.Animal experiments demonstrated that Xiaoer Zhixiao Pingchuan Granules significantly alleviated ovalbumin-induced airway inflammation and goblet cell hyperplasia,while downregulating the expression of key proteins in the PI3K-Akt signaling pathway(P<0.05).Conclusion The therapeutic efficacy of Xiaoer Zhixiao Pingchuan Granules in pediatric asthma involves a multi-pathway and multi-target mechanism,with the PI3K-Akt signaling pathway emerging as a potential key molecular target.
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Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ
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Aim To explore the effect of oxaliplatin combined with epidermal growth factor receptor tyrosine kinase inhibitor AG1478 on autophagy in non-small cell lung cancer H1975 cells. Methods H1975 cells were cultured in vitro using gradient concentrations of AG1478 (0, 5, 10, 15, 20, 25, 30, 35, 40 jjimol • IT
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OBJECTIVE To explore the mechanism of Compound lizard powder reducing cisplatin resistance in gastric cancer by regulating glycolytic activity based on phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. METHODS Human gastric cancer MKN45 and MKN45/DDP (cisplatin-resistant) cells were cultured in vitro and intervened with different mass concentrations of cisplatin (0.1, 0.2, 0.4, 0.8, 1.6, 3.2 μg/mL) to detect the survival rate, half inhibitory concentration (IC50) and drug resistance index. MKN45/DDP cells were inoculated subcutaneously in the right anterior axilla of nude mice to prepare a transplanted tumor model of gastric cancer. After successful modeling, they were randomly divided into model group, cisplatin group (0.002 g/kg), Compound lizard powder group (2.8 g/kg) and combination group (the same dose as each single drug group), with 8 nude mice in each group. Each administration group was given relevant solution, twice a week (cisplatin, i.p.) or twice a day (Compound lizard powder, i. g.), for 4 consecutive weeks. During the experiment, the body weight of nude mice was monitored, and tumor volume and inhibitory rate of tumor were calculated. The levels of inflammatory factors (tumor necrosis factor- α, interleukin-6) in tumor tissue, the mRNA and protein expressions of multidrug resistance-associated protein 1 (MRP1), P-glycoprotein (P-gp), glucose transporter-1 (GLUT1) and lactate dehydrogenase A (LDHA), as well as the protein expressions of PI3K, phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), hexokinase-2 (HK2) and pyruvate kinase M2 (PKM2) were all detected. RESULTS With the intervention of different concentrations of cisplatin, the survival rate of MKN45/DDP-resistant cells was significantly higher than that of MKN45 parent cells (P<0.05). IC50 value of MKN45/DDP and MKN45 cells were(1.052 0±0.221 9) and (0.372 1±0.238 0)μg/mL, and the drug resistant index was 2.827. Compared with the model group, cisplatin group, Compound lizard powder group and combination group all had certain inhibitory effects on the tumor growth in nude mice; the inhibitory rates of tumor increased significantly (P<0.05); the levels of inflammatory factors, the mRNA and protein expressions of MRP1, P-gp, GLUT1 and LDHA (except for cisplatin group), the phosphorylation levels of PI3K and Akt protein (except for cisplatin group) as well as the protein expressions of HK2 and PKM2 were decreased significantly, while the combination group was significantly better than the cisplatin group (P<0.05). CONCLUSIONS Compound lizard powder may inhibit tumor growth in transplanted tumor model nude mice with gastric cancer-resistant cells by reducing the secretion of tumor-related inflammatory factors, inhibiting the expression of glycolysis, drug resistance-related proteins and genes, inhibiting the activation of the PI3K/Akt signaling pathway, thus having a certain effect of enhancing cisplatin efficacy and reversing drug resistance.
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Renal interstitial fibrosis(RIF) is the main pathological manifestation of chronic kidney disease. Due to the complexity of the mechanism, there is no specific treatment for RIF in clinical practice. The abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway and the activation of downstream target genes are key drivers of RIF induction and progression. Traditional Chinese medicine has the characteristics of precise efficacy and minimal toxic side effects, and the occurrence and development of RIF can be regulated by multiple targets and mutual coordination. This review focuses on the PI3K/Akt signaling pathway and summarizes the potential targets and regulatory mechanisms of traditional Chinese medicine in the treatment of RIF. It is found that various effective ingredients (such as sinomenine, mangiferin, coumarin derivates from Hydrangea paniculata, etc.) and formulas (such as Fushengong decoction, Qi-Bang-Yi-Shen formula, etc.) of traditional Chinese medicine can inhibit fibroblast proliferation, improve inflammation and oxidative stress, maintain mitochondrial stability, and slow down ferroptosis through this pathway, thereby delaying the occurrence and progression of RIF.
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AIM: To study the effect and mechanism of Di'ao Xinxuekang (DXXK) on insulin resistance in nonalcoholic steatohepatitis (NASH) mice. METHODS: C57BL/6J mice were randomly divided into normal group and model group. After 16 weeks of high-fat diet, the model group was randomly divided into model group and Pioglitazone group (6.0 mg · kg
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OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non- small cell lung cancer A549 cells. METHODS Using A549 cells as subjects, the effects of different concentrations of gracillin (0.25, 0.5, 1, 2, 4 μmol/L) on the proliferation of cells were detected by CCK-8 after being treated for different time (12, 24, 48 h). Compared with the control group without medication, the effect of gracillin (2 μmol/L) on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure. The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin (0.25, 0.5, 1, 2 μmol/L) for 24 h. Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A(FAM102A), the expressions of autophagy-related proteins [p62, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)], and the expressions of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway-related proteins in A549 cells after being treated with gracillin (0.25, 0.5, 1 and 2 μmol/L) for 24 h. RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. The IC50 was 2.55 μmol/L at 24 h. After 24 h of gracillin treatment, autophagosomes with bilayer membrane structure were found in the cell cytoplasm, and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious, representing an increasing trend as drug concentration. Compared with the control group, mRNA and protein expressions of FAM102A (0.5, 1, 2 μmol/L groups), protein expression of Beclin-1 (1, 2 μmol/L groups) and LC3B-Ⅱ/LC3B-Ⅰ ratio (2 μmol/L group) were significantly increased in different concentrations of gracillin groups, while the protein expression of p62 (1, 2 μmol/L groups), and the protein phosphorylations of Akt (1, 2 μmol/L groups) and PI3K (2 μmol/L group) were all decreased significantly (P<0.05 or P<0.01). CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway, thus inhibiting cell proliferation.
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ObjectiveTo analyze the antidepressant quality markers(Q-Marker) of Bupleuri Radix(BP) before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS), multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP, and principal component analysis(PCA) orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to identify the differential components in BP that changed significantly before and after vinegar-processing, which were regarded as candidate quality markers(Q-Marker). Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology, and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group, model group, fluoxetine group(2.67 mg·kg-1) and total saponin group(0.72 mg·kg-1), except the blank group, rats in the other groups were subjected to chronic unpredictable mild stress(CUMS). Three weeks after the start of modeling, rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks, and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling, 21 days and 28 days after administration, body mass weighing, sucrose preference test and open field test were performed on each group . After 28 days of administration, real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), mammalian target of rapamycin(mTOR), glycogen synthase kinase-3β(GSK-3β), forkhead box transcription factor O3a(FoxO3a) and β-catenin in hippocampal tissues of rats in each group, while protein expression levels of PI3K, Akt, mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing, and 9 components such as saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network, saikosaponin A, saikosaponin B1, saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests, after 28 d of administration, compared with the blank group, the body mass, sucrose preference index and open field total score of rats in model group, fluoxetine group and total saponin group decreased significantly(P<0.01). Compared with the model group, the body mass, sucrose preference index and open field total score in total saponin group increased significantly(P<0.01). Compared with the blank group, mRNA expression levels of PI3K, Akt, mTOR and β-catenin in hippocampus of rats in the model group decreased significantly(P<0.05), while mRNA expression levels of GSK-3β and FoxO3a increased significantly(P<0.05). Compared with the model group, mRNA expression levels of PI3K, Akt, mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly(P<0.05), while mRNA expression levels of GSK-3β and FoxO3a decreased significantly(P<0.05). Compared with the blank group, the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly(P<0.01), while the protein expression levels of PI3K and FoxO3a increased significantly(P<0.01). Compared with the model group, the expression level of Akt in hippocampus of the total saponin group increased significantly(P<0.01), the mTOR expression level was increased but not statistically significant, while the protein expression levels of PI3K and FoxO3a decreased significantly(P<0.01). ConclusionThe chemical constituents of BP changed greatly after vinegar-processing, and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis, pharmacodynamics, network pharmacology and signaling pathway, which provided a reference for further research on quality control, pharmacodynamic substance basis and processing mechanism of BP.
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OBJECTIVE To explore the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis (HF). METHODS Intragastrical administration of carbon tetrachloride was given to induce HF model. By observing the pathological changes in liver tissue, mRNA and protein expressions of HF indexes [α-smooth muscle actin (α-SMA), collagen type Ⅰ] and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway-related factors were detected, and the improvement effects and possible mechanism of low-dose, medium-dose and high-dose (50, 100, 200 mg/kg) of alcoholic extract from S. comosa on HF model rats were investigated. Drug-containing serum was prepared by intragastrical administration of alcoholic extract from S. comosa at a concentration of 1 800 mg/(kg·d) (calculated by the amount of raw material). The effects of drug- containing serum of alcoholic extract from S. comosa on the expression of miRNA-21 were observed through the intervention of HSC-T6 cells with low, medium and high concentrations of drug-containing serum of alcoholic extract from S. comosa (diluted to 10%, 15%, 20%). miRNA-21 mimics or inhibitors were used to transfect HSC-T6 cells, and the mRNA and protein expressions of factors related to the PI3K/Akt signaling pathway were detected. RESULTS The results of in vivo experiments showed that low, medium and high doses of alcoholic extract from S. comosa significantly ameliorated the histopathological changes in liver tissue of HF rats, and the percentage of collagen was significantly reduced (P<0.01); mRNA and protein expressions of the indicators related to HF as well as PI3K and Akt were significantly reduced (P<0.01), and mRNA and protein expressions of phosphatase and tensin homolog deleted on chromosome ten (PTEN) were increased in liver tissue of rats (P<0.01). The results of in vitro experiments showed that drug-containing serum of alcoholic extract from S. comosa significantly inhibited the expression of miRNA-21 at low, medium and high concentrations (P<0.01); whereas after transfection with miRNA-21 mimics, it was found that miRNA-21 mimics significantly increased mRNA and protein expressions of PI3K and Akt (P<0.01), while significantly decreased mRNA and protein expressions of PTEN (P<0.01); after transfection with miRNA-21 inhibitor, the changes of above indexes were opposite to the above results (P<0.01). CONCLUSIONS Alcoholic extracts of S. comosa may inhibit the PI3K/Akt signaling pathway by affecting the expression of miRNA-21, so as to achieve the effect of anti-hepatic fibrosis.
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AIM To observe the protective effects of Qigu Capsule-medicated serum on C2C12 myotube cell atrophy induced by dexamethasone.METHODS The Qigu Capsule-medicated serum was prepared.C2C12 cells cultured in vitro were divided into the normal control group,the dexamethasone group,the 10%blank serum group and the 10%Qigu Capsule-medicated serum group for 48 h,corresponding drug treatment,followed by 24 h culture with 10 μmol/L dexamethasone except the control group,and had cell viability detection by CCK-8 method.With their myotube cells intervened accordingly by the aforementioned regime following 6-7 days differentiation with 2%horse serum induction,the C2C12 cells had their myotube cells diameter measured;and the expressions of proteins related to MyHC,MuRF-1,Atrogin-1 and PI3K/Akt signaling pathway detected by Western blot.The impact of PI3K inhibitor LY294002 on the diameter of myotube cells and the expression of MuRF-1,Atorgin-1 and PI3K/Akt signaling pathway related proteins were detected as well.RESULTS Compared with the normal control group,the dexamethasone group and the blank serum group were observed with decreased viability of C2C12 cells(P<0.01),decreased diameter of myotube cells(P<0.01),increased protein expressions of MuRF-1 and Atrogin-1(P<0.01),and decreased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.01).Compared with the dexamethasone group,the Qigu Capsule-medicated serum group showed increased viability of C2C12 cells(P<0.01);increased diameter of myotube cells(P<0.01);decreased protein expressions of MuRF-1 and Atrogin-1(P<0.01);and increased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).The intervention of LY294002 upon the Qigu Capsule-medicated serum group offset the anti-muscular tube atrophy effect(P<0.01),increased the protein expressions of MuRF-1 and Atorgin-1(P<0.01),and decreased the phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).CONCLUSION The Qigu Capsule-medicated serum can alleviate the atrophy of C2C12 myotubes induced by dexamethasone,and its mechanism may be related to the activation of PI3K/Akt signaling pathway.
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Chikusetsusaponin IVa (CsIVa) is a natural active monomer of triterpene saponins in the Chinese herbal medicine of Panax japonicus, which has anti-inflammatory, anti-tumor and other effects. However, its function and mechanism in triple negative breast cancer (TNBC) remain unclear. This study investigated the inhibitory effect and mechanisms of CsIVa on the proliferation of triple negative breast cancer cell line MDA-MB-231. In this study, we found that CsIVa could significantly inhibit the proliferation of MDA-MB-231 cells and eliminate its potential toxic effect on normal breast cells (MCF-10A). The transcriptome sequencing results showed that the inhibition of proliferation of MDA-MB-231 cells by CsIVa was closely related to cell cycle and the pathway regulating cell cycle. Further studies confirmed that CsIVa blocked the cell cycle in G2/M phase by down-regulating the expression of cyclin dependent kinase 1 (CDK1), cyclin B1 and up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21). Moreover, CsIVa can block cell cycle through inhibiting PI3K/AKT signal pathway. In conclusion, CsIVa regulates the expression of cell cycle related proteins (p21, CDK1, cyclin B1) via inhibiting the activity of PI3K/AKT signaling pathway, blocks TNBC cell cycle, and thus exerts its anti-tumor activity.
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OBJECTIVE@#To investigate the mechanism of the effect of Astragalus membranaceus (A. membranaceus) on lung adenocarcinoma at the molecular level to elucidate the specific targets according to the network pharmacology approach.@*METHODS@#The active components of A. membranaceus and their potential targets were collected from the Traditional Chinese Medicine Systems Pharmacology Database. Lung adenocarcinoma-associated genes were acquired based on GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, and Therapeutic Targets databases. The PI3K/AKT signaling pathway-related genes were obtained using Reactome portal. Networks of "ingredient-target" and "ingredient-target-pathway-disease" were constructed using the Cytoscape3.6.0 software. The relationships among targets were analyzed according protein-protein interaction (PPI) network. Finally, molecular docking was applied to construct the binding conformation between active ingredients and core targets. Cell counting kit 8 (CCK8) and Western blot assays were performed to determine the mechanism of the key ingredient of A. membranaceus.@*RESULTS@#A total of 20 active components and their 329 targets, and 7,501 lung adenocarcinoma-related genes and 130 PI3K/AKT signaling pathway-related genes were obtained. According to Venn diagram and PPI network analysis, 2 mainly active ingredients, including kaempferol and quercetin, and 6 core targets, including TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR, were identified. The two important active ingredients of A. membranaceus, kaempferol and quercetin, exert the therapeutic effect in lung adenocarcinoma partly by acting on the 6 core targets (TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR) of PI3K/AKT signaling pathway. Expressions of potential targets in lung adenocarcinoma and normal samples were analyzed by using UALCAN portal and found that ERBB2 was overexpressed in lung adenocarcinoma tissues and upregulation of it correlated with clinicopathological characteristics. Finally, quercetin repressed viabilities of lung adenocarcinoma cells by targeting ERBB2 on PI3K/AKT signaling confirmed by CCK8 and Western blot.@*CONCLUSION@#Our finding unraveled that an active ingredient of A. membranaceus, quercetin, significantly inhibited the lung adenocarcinoma cells proliferation by repressing ERBB2 level and inactivating the PI3K/AKT signaling pathway.
Subject(s)
Humans , Astragalus propinquus , Kaempferols , Network Pharmacology , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Epidermal Growth Factor , Molecular Docking Simulation , Quercetin , Adenocarcinoma of Lung , Lung Neoplasms , Signal Transduction , ErbB Receptors , Drugs, Chinese HerbalABSTRACT
Objective To investigate the preventive effect of schisandrin(SCH)on fetal neural tube defects(NTDs)of mice and its mechanism.Methods C57BL/6 mice were mated with female and male at a ratio of 2:1.Pregnant female mice with vaginal plug after mating were randomly divided into control group,model group,SCH group,and folic acid group,with 9 mice in each group.The NTDs fetal mice model was induced by intraperitoneal injection of all-trans retinoic acid(atRA)(7.5 mg/kg)on embryonic day 7.5(E 7.5 d).During E 0.5 d-E 11.5 d,pregnant rats in folic acid group were given folic acid[61.0 μg/(kg·d)]by gavage once a day,and pregnant rats in SCH group were given SCH[8.0 mg/(kg·d)]by gavage once a day.Fetal mice were removed by cesarean section on E 11.5 d.PC12 cells were divided into control group,model group and SCH group.PC12 cells were treated with atRA(20 μmol/L)for 12 hours to establish cell damage model in model group,and treated with SCH(2.5 μmol/L)for 24 hours in SCH group.Fetuses were identified NTDs by stereoscopic microscopy.HE staining was used to observe the closure of the neural tube.The expression levels of p-PI3K,Akt and p-Akt molecules in PI3K/Akt signaling pathway were detected by Western Blotting.Results Compared with control group,the incidence of NTDs was significantly increased in mice of model group(P<0.01);compared with model group,the incidence of NTDs was decreased in folic acid group and SCH group(P<0.01);compared with folic acid group,SCH group had a lower incidence of NTDs(P<0.01).Western Blotting results showed that compared with control group,the expression of p-PI3K and p-Akt protein in fetal tissues of model group was significantly decreased(P<0.01,P<0.05);compared with model group,there was no significant difference in expression of p-PI3K and p-Akt in fetal tissues of folic acid group(P>0.05),while the expression of p-PI3K and p-Akt protein in SCH group was significantly higher(P<0.05).Compared with control group,PC12 cells in model group showed lower expression levels of p-PI3K and p-Akt(P<0.05);compared with model group,PC12 cells in SCH group showed higher expression levels of p-PI3K and p-Akt(P<0.05).Conclusions SCH can reduce the incidence of atRA-induced NTDs in fetal mice,and its preventive effect is better than folic acid,which may be related to the activation of the PI3K/Akt signaling pathway.
ABSTRACT
Objective·To investigate the effect of F-box only protein 38(FBXO38)on the ocular melanoma proliferation and the potential regulatory pathway.Methods·Human skin cutaneous melanoma A375 and human uveal melanoma OMM2.3 cell lines with FBXO38 knockdown and overexpression were constructed by FBXO38 short hairpin RNA(shRNA)and FBXO38 overexpression plasmids respectively.Knockdown and overexpression efficiency of FBXO38 at transcription and protein levels were verified by using quantitative real-time PCR(qRT-PCR)and Western blotting.The effects of FBXO38 on melanoma cell proliferation were detected through clonal formation assay,BrdU immunofluorescence staining and CCK8 cell proliferation assay.By using The Cancer Genome Atlas(TCGA)database,differentially expressed genes were analyzed in the high and low expression groups of FBXO38.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment was performed to reveal the signaling pathways associated with FBXO38.CCK8 cell proliferation assay was used to detect the inhibition rates of the signaling pathway inhibitors on cells with different FBXO38 expression levels.qRT-PCR and Western blotting were used to detect whether the signaling pathway was activated after knocking down FBXO38.Results·qRT-PCR and Western blotting verified that mRNA and protein expression levels of FBXO38 in FBXO38 knockdown A375 and OMM2.3 cell lines decreased compared with the control group,while the expression levels of FBXO38 in the overexpression cell lines increased compared with wild type group(P<0.05).Clonal formation assay,BrdU immunofluorescence staining and CCK8 cell proliferation assay showed that FBXO38 knockdown significantly enhanced the proliferation of A375 and OMM2.3 cells(P<0.05),while overexpression of FBXO38 inhibited melanoma cell proliferation(P<0.05).Enrichment analysis showed that in skin cutaneous melanoma and uveal melanoma,FBXO38 expression influenced the phosphoinositide 3-kinase/protein kinase B(PI3K-Akt)pathway activation.Compared with those in the control group,the inhibition rates of P13K inhibitor LY294002 and mTOR1 inhibitor Everolimus in the FBXO38 knockdown group significantly improved(P<0.05),while their inhibition rates of the overexpression group significantly decreased compared with those of control cells(P<0.05).Western blotting results showed that after knocking down FBXO38,expression levels of PTEN,P21 and P53 proteins decreased,while expression level of MDM2 protein increased.The qRT-PCR results showed a significant decrease in P53 transcription level(P<0.05)and a significant increase in MDM2 transcription level in FBXO38 knockdown cells(P<0.05).Conclusion·FBXO38 plays a role in regulating the proliferation of ocular melanoma,and this regulatory effect is related to the PI3K-Akt signaling pathway.