Effect of South African Herb Hoodia gordonii on Liver Glucolipid Metabolism and PI3K/Akt/FoxO1 Signalling Pathway in db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the South African herb Hoodia gordonii （HG） on glucolipid metabolism in diabetic db/db mice and explore the possible mechanisms of HG on the liver of db/db mice based on the phosphoinositide-3 kinase （PI3K）/protein kinase B （Akt）/factor forkhead protein O1 （FoxO1） signaling pathway. MethodA total of 30 db/db mice were randomly divided into five groups according to fasting blood glucose： model group， metformin group （0.195 g·kg-1）， and low dose （0.39 g·kg-1）， medium dose （0.78 g·kg-1）， and high dose （1.56 g·kg-1） HG groups， with six m/m mice in each group， and another six m/m mice were set as normal group. The mice in the normal and model groups were given saline of 9 mL·kg-1 by gavage. Body weight， water intake， and fasting blood glucose of the mice in each group were measured weekly. After six weeks of continuous administration， serum insulin （FINS）， low-density lipoprotein cholesterol （LDL）， total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， urea， and creatinine （CREA） were measured， and liver sections were embedded and stained with hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and oil red O. Protein expression of PI3K p85， p-Akt， and p-FoxO1 in liver was detected by immunohistochemistry. The mRNA expression of PI3K， Akt， and FoxO1 in liver tissue was detected by real-time polymerase chain reaction （Real-time PCR）. ResultAfter six weeks of administration intervention， it was found that fasting blood glucose was significantly downregulated in mice in the three HG groups （P<0.05）. The level of islet resistance index was significantly reduced in both the low and medium dose HG groups （P<0.05）. The expression levels of TC， TG， and LDL were reduced in all HG groups （P<0.05， P<0.01）. Pathologically， HG could alleviate hepatocyte steatosis， reduce the volume and content of lipid droplets in liver， and increase the distribution of glycogen granules in liver to some extent in mice. Immunohistochemical assays revealed that PI3K p85 protein expression was significantly increased in the low， medium， and high dose HG groups compared with the model group （P<0.01）. p-Akt protein expression was significantly increased in the medium and high dose HG groups （P<0.05， P<0.01）. p-FoxO1 protein expression was significantly increased in the low， medium， and high dose HG groups （P<0.05， P<0.01）. Compared with the model group， PI3K mRNA was increased in low dose， medium dose， and high dose HG groups （P<0.05）， and Akt mRNA was increased in high dose HG group （P<0.05）. FoxO1 mRNA was decreased in low dose， medium dose， and high dose HG groups （P<0.05）. ConclusionHG can ameliorate the disorder of glucolipid metabolism in db/db mice， which may be related to its activation of the hepatic PI3K/Akt/FoxO1 signaling pathway.

Fangji Fuling Decoction Alleviates Sepsis by Blocking MAPK14/FOXO3A Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments.@*METHODS@#A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed.@*RESULTS@#FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05).@*CONCLUSION@#FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.

Effects of Ginkgo biloba extract on renal injury in rats with experimental renal failure through miR-145/FOXO1 axis / 中国临床药理学与治疗学

ABSTARCT AIM: To investigate the effect of Ginkgo biloba extract (GBE) on kidney injury in rats with chronic renal failure (CRF) and its potential molecular mechanism. METHODS: SD rats were given 5/6 nephrectomy to construct CRF models and divided into model group, GBE group (100 mg /kg), GBE+ Agomir-NC group, and GBE+Agomir-145 group, 12 per group; another 12 were selected as the sham group, with only the kidney exposed and no nephrectomy. Rats in the GBE group were given GBE 100 mg/kg gavage daily, once a day, for 4 consecutive weeks; rats in the GBE+Agomir-NC group and GBE+Agomir-145 group were given GBE 100 mg/kg gavage daily, and then Agomir-NC and Agomir-145 were injected via the tail vein every 3 days for 4 weeks; the sham group and the model group were given the same amount of normal saline by gavage and injection through the tail vein respectively. The general state of the rat was observed, and the renal function indicators [24 h urine microalbumin (24 h UAlb), blood urea nitrogen (BUN), blood creatinine (SCr)] and oxidative stress indicators [malonaldehyde (MDA), Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)] were detected, Masson staining was used to observe the fibrosis of kidney tissue, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expression levels of microRNA-145 (miR-145), transforming growth factor - β1 (TGF - β1) and forkhead box O1 (FOXO1) in renal tissue, Western blot was used to detect the protein levels of TGF - β1 and FOXO1 in kidney tissue. RESULTS: The general state of CRF rats improved significantly after GBE intervention, the body weight, renal tissue SOD and GSH-Px activities, and FOXO1 mRNA and protein levels were significantly higher than those in the model group (P<0.05); the 24 h UAlb, serum BUN, SCr and renal tissue MDA levels, the relative area of renal interstitial fibrosis, and renal tissue miR-145, TGF - β1 mRNA and protein levels were significantly lower than those in the model group (P<0.05); and on the basis of GBE intervention, up-regulating the expression of miR-145 could significantly weaken the protective effect of GBE on renal injury in CRF rats (P<0.05). CONCLUSION: GBE can alleviate kidney damage in CRF rats, and its mechanism of action may be related to down-regulation of miR-145, up-regulation of FOXO1 expression, and inhibition of renal fibrosis.

Zhizhu Decoction Alleviates Intestinal Barrier Damage via Regulating SIRT1/FoxO1 Signaling Pathway in Slow Transit Constipation Model Mice / 中国结合医学杂志

OBJECTIVE@#To explore the possible effects and mechanism of Zhizhu Decoction (ZZD) on the pathophysiology of slow transit constipation (STC).@*METHODS@#A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table, including control, STC model (model), positive control, and low-, medium- and high-doses ZZD treatment groups (5, 10, 20 g/kg, namely L, M-, and H-ZZD, respectively), 9 mice in each group. Following 2-week treatment, intestinal transport rate (ITR) and fecal water content were determined, and blood and colon tissue samples were collected. Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells. To determine intestinal permeability, serum levels of lipopolysaccharide (LPS), low-density lipoprotein (LDL) and mannose were measured using enzyme-linked immunosorbent assay (ELISA). Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1 (ZO-1), claudin-1, occludin and recombinant mucin 2 (MUC2). The mRNA expression levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-4, IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction. Colon indexes of oxidative stress were measured by ELISA, and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1 (SIRT1/FoxO1) antioxidant signaling pathway were detected by Western blot.@*RESULTS@#Compared with the model group, ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups (P<0.01). Additionally, ZZD treatment notably increased the thickness of mucosal and muscular tissue, elevated the number of goblet cells in the colon of STC mice, reduced the secretion levels of LPS, LDL and mannose, and upregulated ZO-1, claudin-1, occludin and MUC2 expressions in the colon in a dose-dependent manner, compared with the model group (P<0.05 or P<0.01). In addition, ZZD significantly attenuated intestinal inflammation and oxidative stress and activated the SIRT1/FoxO1 signaling pathway (P<0.05 or P<0.01).@*CONCLUSION@#ZZD exhibited beneficial effects on the intestinal system of STC mice and alleviated intestinal inflammation and oxidative stress via activating SIRT1/FoxO1 antioxidant signaling pathway in the colon.

Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Gegen Qinliantang （GQT） in improving ectopic lipid accumulation in the liver of db/db mice with type 2 diabetes mellitus （T2DM） by regulating the adenosine monophosphate-activated protein kinase （AMPK）-forkhead box O3a （FoxO3a） autophagy axis， to provide a scientific basis for clarifying the hypoglycemic mechanism of GQT and its clinical application. MethodSeventy-five spontaneous T2DM db/db mice and 15 normal db/m mice were selected and maintained on a regular diet for one week， followed by the measurement of blood glucose. They were then randomly divided into six groups， with 15 mice in each group， including normal group （0.2 g·kg-1 saline）， metformin group （0.2 g·kg-1）， high-， medium， and low-dose GQT group （31.9， 19.1， 6.9 g·kg-1）， and model group （0.2 g·kg-1 saline）. The mice were orally administered the corresponding drugs once daily for 12 weeks. Fasting blood glucose （FBG） and glycated hemoglobin （HbA1c） were detected. Fasting insulin （FINS） and free fatty acid （FFA） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Pathological changes in liver tissues were observed by hematoxylin-eosin （HE） staining. The protein expression levels of phosphorylated （p）-AMPK， p-FoxO3a， and autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ） and p62 were detected using Western blot. Immunofluorescence was used to detect the expression of hypoxia-inducible factor-1α （HIF-1α） in liver tissues. Real-time polymerase chain reaction （Real-time PCR） was performed to detect the mRNA expression of AMPK， FoxO3a， and LC3 in liver tissues. ResultCompared with the normal group， the model group showed pathological changes in liver tissues， increased FBG， HbA1c， FINS， and FFA levels （P<0.01）， increased protein expression levels of p-AMPK， p62， and HIF-1α， decreased protein expression levels of p-FoxO3a and LC3Ⅱ in liver tissues （P<0.01）， decreased mRNA expression of AMPK， and increased expression of FoxO3a （P<0.01）. Compared with the model group， the treatment groups showed relieved liver tissue lesions and decreased FBG， HbA1c， FINS， and FFA levels （P<0.01）. The expression of p-AMPK， p62， and HIF-1α increased， while the expression of p-FoxO3a showed a dose-dependent decrease in the high-dose GQT group. The expression of LC3Ⅱ increased in the metformin group and the high-dose GQT group （P<0.01）. The mRNA expression of AMPK showed a dose-dependent increase， and the expression of FoxO3a showed a dose-dependent decrease in the treatment groups （P<0.01）. ConclusionGQT can improve ectopic lipid accumulation in the liver of T2DM db/db mice， which may be related to the regulation of the AMPK-FoxO3a autophagy axis.

Association of FOXO3 polymorphism (rs3800231) and clinical subphenotypes of beta thalassemic individuals

Abstract Introduction Studies have shown that the loss of the FOXO3 transcriptional function is involved in the pathophysiology of some chronic erythroid disorders, including beta-thalassemia (β-thal). Therefore, the single nucleotide polymorphism (SNP) rs3800231 (35-2764A > G) could contribute to alterations in its transcriptional activity, acting as a modifier of β-thal phenotypic manifestations. Objective and method In order to better understand the genotypic and/or allelic distributions among β-thal patients, we evaluated 83 β-thal heterozygous and 20 homozygous, compared to 117 individuals without hemoglobinopathies (control group). Additionally, we verified any influence of the FOXO3 polymorphism on clinical manifestations among β-thal homozygotes. Results We obtained higher frequencies of the wild-type homozygous (AA) and the wild-type allele (A) in the β-thal group (p< 0.0001 and p= 0.00014, respectively). The most common clinical manifestations found among β-thal homozygotes were iron overload (90%), splenomegaly (65%) and bone complications (35%), e.g., osteopenia/osteoporosis. We observed that close to 80% of the patients presenting such manifestations had the genotype AA. However, we did not find any significant involvement of the FOXO3 polymorphism in clinical manifestation occurrences. Conclusion Thus, we concluded that the SNP rs3800231 did not play a significant role as a modifier of the clinical manifestations observed in the β-thal homozygotes studied.

Dendrocalamus latiflorus and its component rutin exhibit glucose-lowering activities by inhibiting hepatic glucose production via AKT activation

The potential medicinal value of Ma bamboo (Dendrocalamus latiflorus), one of the most popular and economically important bamboo species in China, has been underestimated. In the present study, we found that D. latiflorus leaf extract (DLE) reduced fasting blood glucose levels, body weight, and low-density lipoprotein cholesterol with low liver toxicity in db/db mice. In addition, gene expression profiling was performed and pathway enrichment analysis showed that DLE affected metabolic pathways. Importantly, DLE activated the AKT signaling pathway and reduced glucose production by downregulating glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) expression. Moreover, network pharmacology analysis identified rutin as an active component in DLE through targeting insulin growth factor 1 receptor (IGF1R), an upstream signaling transducer of AKT. Due to its hypoglycemic effects and low toxicity, DLE may be considered an adjuvant treatment option for type 2 diabetes patients.

Effect of Trichosanthis Radix-Polygonati Rhizoma on Glucose and Lipid Metabolism and Hepatic Insulin Resistance in KKAy Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the water extracts of Trichosanthis Radix-Polygonati Rhizoma at different ratios on glucose and lipid metabolism in KKAy mice with spontaneous type 2 diabetes and explore the mechanism of the extract in alleviating insulin resistance based on phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead box O1 （FoxO1） signaling pathway. MethodThe 8-week-old C57BL/6J male mice were taken as the normal control group， and KKAy male mice of the same age were randomly assigned into a model group， a metformin group， Trichosanthis Radix-Polygonati Rhizoma groups at the ratios of 1∶1 （Trichosanthis Radix 30 g， Polygonati Rhizoma 30 g）， 1∶3 （Trichosanthis Radix 15 g， Polygonati Rhizoma 45 g）， and 1∶5 （Trichosanthis Radix 10 g， Polygonati Rhizoma 50 g） according to blood glucose level and body weight， with 6 mice in each group. The administration lasted for 8 weeks， and the body weight （BW） and fasting blood glucose （FBG） of mice were recorded at the same time points of the 2nd， 4th， 6th， and 8th weeks， respectively. Oral glucose tolerance test （OGTT） was performed at the 7th week. After drug administration， the serum levels of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， and fasting insulin （FINS） were measured， and homeostasis model assessment-insulin resistance （HOMA-IR） index was calculated. The liver tissue samples were stained with hematylin-eosin （HE） and periodic acid-Schiff （PAS） for observation of the fat distribution and glycogen content. The protein levels of PI3K， Akt， p-Akt， FoxO1， and p-FoxO1 in the liver were determined by Western blot. ResultCompared with the normal group， the model group showed increased food intake， FBG， glucose tolerance， FINS， HOMA-IR， TC， TG， and LDL-C （P<0.01）， and down-regulated protein levels of PI3K， Akt， phosphorylaison （p）-Akt， FoxO1， and p-FoxO1 in the liver （P<0.01）. Compared with the model group， Trichosanthis Radix-Polygonati Rhizoma lowered FBG and HOMA-IR （P<0.05， P<0.01）. In particular， the combination at the ratio of 1∶3 showed the best performance （P<0.01） comparable to metformin. Furthermore， Trichosanthis Radix-Polygonati Rhizoma at different ratios lowered blood glucose at different time points of OGTT （P<0.05） and TC and LDL-C （P<0.01）. Additionally， the combination at the ratio of 1∶3 reduced TG （P<0.01）. The liver of mice in the drug administration groups showed regular morphology， with few lipid droplets and rich glycogen. Western blot showed that Trichosanthis Radix-Polygonati Rhizoma up-regulated the protein levels of PI3K and p-Akt， down-regulated the protein level of FoxO1， and up-regulated the protein level of p-FoxO1 （P<0.05）. ConclusionTrichosanthis Radix-Polygonati Rhizoma， especially at the ratio of 1∶3， lowered the FBG， TC， LDL-C， and HOMA-IR index， promoted liver glycogen synthesis， and reduced steatosis in KKAy mice， which may be related to the regulation of PI3K/Akt/FoxO1 signaling pathway in the liver.

Effects of hyperbaric oxygen on the blood-brain barrier via the SIRT1/FoxO1 signaling pathway after cerebral ischemia and reperfusion / 中华物理医学与康复杂志

Objective:To explore the effect of hyperbaric oxygen (HBO) on the blood-brain barrier via the silent information regulator 1 (SIRT1)/Forkhead box O1(FoxO1) signaling pathway after cerebral ischemia and reperfusion using a rat model.Methods:Forty Wistar rats were randomly assigned into sham, cerebral ischemia-reperfusion (CIR), CIR+ HBO and CIR+ HBO+ EX527 groups, each of 10. The cerebral ischemia-reperfusion model was established in all groups except the sham group by right middle cerebral artery occlusion using the modified thread-occlusion method. The sham group was not ligated. Both the CIR+ HBO and CIR+ HBO+ EX527 groups were given HBO 1, 9, 21, 45 and 69 hours after the reperfusion. The CIR+ HBO+ EX527 group was additionally injected with 5mg/kg of EX527(a SIRT1inhibitor) peritoneally 4, 12, 24, 48 and 72 hours after the reperfusion. Then 2% Evens blue (EB) was injected into the tail vein an hour before the rats were sacrificed. The content of EB and the expression of SIRT1, FoxO1, ZO-1, Occludin, Claudin-5 mRNA and their proteins were determined using spectrophotometry, reverse transcription-polymerase chain reactions and Western blotting.Results:The average EB content of the hippocampal brain tissue from the CIR, CIR+ HBO and CIR+ HBO+ EX527 rats was significantly greater than the Sham group′s average 72h after reperfusion. The average expression of SIRT1, FoxO1, ZO-1, Occludin and Claudin-5 mRNA and their proteins was significantly lower, with the CIR + HBO + EX 527 group′s average significantly lower than that of the CIR+ HBO group.Conclusions:HBO can increase the expression of tight junction protein via the SIRT1/FoxO1 pathway. It helps to protect the blood-brain barrier in CIR injury situations.

Effect of Liuwei Dihuangwan on Cognitive Ability of SAMP8 Mice by Regulating Autophagy Through PI3K/Akt/FoxO3a Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Liuwei Dihuangwan on memory function of senescence-accelerated mouse prone 8 （SAMP8） mice by regulating autophagy through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead box O3a （FoxO3a） pathway. MethodSix male senescence-accelerated mouse resistant 1 （SAMR1） mice of SPF grade aging 6 months were assigned to a normal group， and 24 male SAMP8 mice of SPF grade aging 6 months were randomly divided into a model group， a donepezil group （0.747 mg·kg-1）， and high- and low-dose Liuwei Dihuangwan groups （2.700 and 1.350 g·kg-1）， with 6 mice in each group. The mice were treated with drugs by gavage for 2 months. Morris water maze was used to detect the learning and memory abilities of mice in each group. Nissl staining was used to observe the neurons in the cortex and hippocampus. The positive expression of microtubule-associated protein 1 light chain 3B （LC3B） in the cortex and hippocampus was detected by immunohistochemistry （IHC）. Western blot was used to detect the protein expression of the mammalian ortholog of yeast ATG6 （Beclin-1）， B cell lymphoma-2 （Bcl-2）， autophagy-related gene 5 （ATG5）， cysteinyl aspartate-specific protease 3 （Caspase-3）， Caspase-9， Akt， p-Akt， FoxO3a， and p-FoxO3a. ResultCompared with the normal group， the model group showed prolonged escape latency （P<0.05，P<0.01）， reduced number of platform crossings and the residence time in the target quadrant （P<0.01）， decreased neurons with reduced volume and dispersed distribution in the cortex and hippocampus， increased positive expression of LC3B （P<0.01）， elevated expression of Beclin-1 and ATG5 in the cortex （P<0.01）， declined Bcl-2 expression （P<0.01）， up-regulated Caspase-3 and Caspase-9 expression （P<0.01）， and decreased expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. Compared with the model group， the donepezil group and the Liuwei Dihuangwan groups showed shortened 3 d escape latency （P<0.05，P<0.01）， increased number of platform crossings （P<0.01）， and prolonged residence time in the target quadrant （P<0.01）. In the donepezil group， the number of neurons in the cortex and hippocampus was increased. In the Liuwei Dihuangwan groups， the number of neurons and Nissl bodies increased with denser distribution and lower degree of cell damage. The positive expression of LC3B in the cortex and hippocampus was decreased in the donepezil group and Liuwei Dihuangwan groups （P<0.01）. The expression of Beclin-1 was decreased in the Liuwei Dihuangwan groups （P<0.01）. The expression of ATG5 was decreased in the donepezil group and the low-dose Liuwei Dihuangwan group （P<0.01）. The donepezil group and the Liuwei Dihuangwan groups showed the increased expression level of Bcl-2 in the cortex （P<0.01）， decreased expression level of Caspase-3 （P<0.01）， reduced expression level of Caspase-9 （P<0.05，P<0.01）， and elevated expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a （P<0.01）. ConclusionLiuwei Dihuangwan can effectively improve the learning and memory abilities of the SAMP8 mice and protect neurons. Its mechanism may be related to the regulation of the PI3K/Akt/FoxO3a signaling pathway， down-regulation of the expression of ATG5， Beclin-1， and LC3B， and the inhibition of apoptosis.

Study on expression relationship of DNA methyltransferase 1 and forkhead box O3a in colon cancer / 国际肿瘤学杂志

Objective:To analyze the relationship between DNA methyltransferase 1 (DNMT1) and forkhead box O3a (FOXO3a) in colon cancer and the diagnostic efficacy of combined detection in predicting the occurrence of colon cancer by detecting the levels of DNMT1 and FOXO3a in serum of colon cancer patients.Methods:A total of 105 patients with colon cancer diagnosed and treated in Xi′an International Medical Center Hospital from September 2019 to September 2020 were selected as the colon cancer group, and 65 patients with colon polyps diagnosed by biopsy during the same period were selected as control group. The levels of DNMT1 and FOXO3a in serum of patients were detected by real-time fluorescence quantitative PCR. Pearson correlation coefficient method was used to analyze the correlation between the levels of DNMT1 and FOXO3a in serum of patients with colon cancer. Subject operating characteristic curve was used to evaluate the diagnostic values of DNMT1 and FOXO3a levels in colon cancer.Results:The serum levels of DNMT1 in the control group and colon cancer group were 0.93±0.28 and 1.34±0.35, compared with the control group, the level of DNMT1 in the colon cancer group was significantly higher, with a statistically significant difference ( t=7.990, P<0.001). The serum levels of FOXO3a were 1.04±0.39 and 0.69±0.18, compared with the control group, the level of FOXO3a in the colon cancer group was significantly lower, with a statistically significant difference ( t=7.940, P=0.001). The serum levels of DNMT1 and FOXO3a in patients with colon cancer were negatively correlated ( r=-0.687, P<0.001). The area under the curve (AUC) of DNMT1 predicting colon cancer was 0.843, the sensitivity was 71.40%, and the specificity was 90.80%. The AUC of FOXO3a predicting colon cancer was 0.812, the sensitivity was 88.60%, and the specificity was 67.70%. The AUC of the two combined predicting colon cancer was 0.859, the sensitivity was 89.50%, and the specificity was 92.30%. Compared with FOXO3a single detection, the predictive value of combined detection of DNMT1 and FOXO3a were higher ( Z=1.982, P=0.047). Conclusion:The level of DNMT1 in the serum of patients with colon cancer is increased, while the level of FOXO3a is decreased. There is a negative correlation between them in the serum of patients with colon cancer. The combined detection of the DNMT1 and FOXO3a can effectively improve the diagnostic value of colon cancer.

The Role of PI3K / AKT / FOXO Signaling Pathway-mediated Autophagy in Diabetes-associated Cognitive Dysfunction / 中国生物化学与分子生物学报

Diabetes‚ a metabolic disease characterized by hyperglycemia‚ can cause central nerve system damage‚ lead to alteration of the neuronal structure and function‚ and consequently induce cognitive dysfunction. Recently‚ diabetes-associated cognitive dysfunction (DACD) and its molecular mechanism have become a research frontier. The phospoinositide 3 kinase/ protein kinase B/ Forkhead box O (PI3K/ PKB/ FOXO) signaling pathway is an important upstream regulatory mechanism for autophagy. Here we review the role of the PI3K/ AKT/ FOXO signaling pathway in the regulation of Gs‚ Bnip3 and Spk2 gene expressions. GS regulates the Gln-mTORC1 pathway and thus activates autophagy; BNIP3 enhances LC3 expression and promotes autophagy. Moreover‚ the AMPK-FOXO3a-mTORC1 signaling pathway is also an important pathway that involved in the regulation of autophagy. These studies suggest that FOXO3a may be a key target for the treatment of DACD. This review aims to provide a theoretical basis and molecular target for the clinical treatment of DACD and it related drug development.

Mechanism of Wenshen Yangxue Prescription Improving Endometrial Receptivity of Aged Female Mice by Regulating PI3K/Akt/FoxO3A Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the effective components， targets， and possible mechanisms of Wenshen Yangxue prescription in improving endometrial receptivity of aged female mice based on network pharmacology and experimental verification. Method:Based on Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM） and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine， the components and targets of Wenshen Yangxue prescription were retrieved， and the targets of ovulatory dysfunctional infertility were collected from the Online Mendelian Inheritance in Man （OMIM） and GeneCards with "anovulatory sterility" and "anovulatory infertility" as keywords. The protein-protein interaction （PPI） network was constructed based on STRING and the core targets of Wenshen Yangxue prescription against ovulatory dysfunctional infertility were screened by Cytoscape， followed by Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment of the core targets in DAVID database. Then， the "medicinal-component-target-pathway" network was established and the core targets were verified by animal experiment. Result:A total of 253 components and 326 targets of Wenshen Yangxue prescription， 819 disease targets， and 74 common targets were screened out. The common targets were mainly involved in the biological processes such as positive regulation of nitric oxide biosynthetic process， positive regulation of cell proliferation， response to estradiol， aging， response to oxidative stress， and angiogenesis. The GO term of response to oxidative stress and five of the top 20 KEGG pathways were analyzed. According to the "medicinal-component-target-biological process/pathway" network， 41 chemical components in 20 medicinals participated in hypoxia inducible factor-1 （HIF-1） signaling pathway， tumor necrosis factor （TNF） signaling pathway， forkhead box O （FOXO） signaling pathway， Toll-like receptor （TLR） signal pathway， and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway by affecting 35 targets. The results of animal experiment showed that the prescription could increase the expression of PI3K， phosphorylated PI3K （p-PI3K）， Akt， phosphorylated Akt （p-Akt）， forkhead box O3A （FoxO3A）， and phosphorylated FoxO3A （p-FoxO3A） in uterus of aged female ICR mice. Conclusion:Wenshen Yangxue prescription interferes with oxidative stress and PI3K/Akt/FoxO3A signaling pathway by influencing Akt1， dual oxidase 2 （DUOX2）， epidermal growth factor receptor （EGFR）， heme oxygenase-1 （HMOX1）， myeloperoxidase （MPO）， and other targets， thereby improving endometrial receptivity of aged female mice.

Effect of Koumine on Proliferation and Apoptosis of Colorectal Cancer Cells via ROS/JNK/FoxO3a Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the effect and underlying mechanism of koumine （Kou） at different concentrations （0， 100， 200， 400 μmol·L^-1） on the proliferation and apoptosis of colorectal cancer HCT-116 cells. Method:After 24 hours of<italic> in vitro</italic> intervention with HCT-116 cells by Kou， cell counting kit-8 （CCK-8） assay was used to detect its effect on cell proliferation. Flow cytometry was used to detect cell cycle， apoptosis， and reactive oxygen species （ROS） expression. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of forkhead box O3a （FoxO3a）. Cells were transfected with small interfering ribonucleic acid （siRNA）. Western blot was employed to detect the protein expression of the FoxO3a target gene. Result:Compared with the conditions in the blank group， Kou treatment reduced the proliferation rate of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01） in a dose-dependent manner， caused cell cycle arrest in the G₀/G₁ phase， and induced the apoptosis of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01）， which was positively correlated with the concentration of Kou. FoxO3a siRNA interference reduced the expression of FoxO3a and its downstream target genes cyclin-dependent kinase inhibitor 1A （p21）， cyclin-dependent kinase inhibitor 1B （p27）， and Bcl-2 interacting mediator of cell death （Bim） （<italic>P</italic><0.01）. Kou treatment induced the activation of c-Jun <italic>N</italic>-terminal kinase （JNK） in HCT116 cells. SP600125 （JNK specific inhibitor） treatment inhibited the Kou-induced FoxO3a activation and the expression of its downstream target genes. <italic>N</italic>-acetyl cysteine （NAC） treatment reduced Kou-induced ROS levels （<italic>P</italic><0.01） and JNK signal activation. The above results were significantly different from those in the blank group （<italic>P</italic><0.01）. Conclusion:Kou can effectively inhibit the proliferation of HCT-116 cells and promote apoptosis， and the mechanism may be related to the regulation of the ROS/JNK/FoxO3a pathway.

miR-182-5p enhances proliferation and inhibits apoptosis of A549 lung cancer cells by targeting fork-head box O3a / 国际肿瘤学杂志

Objective To evaluate the effect of microRNA-182-5p (miR-182-5p) on proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells by targeting forkhead box O3a (FOXO3a).Methods The difference of miR-182-Sp expression between human normal lung epithelial cells BEAS-2B and NSCLC cells A549 was compared.The A549 cells were chosen,and miR-182-Sp mimic (miR-182-Sp mimic group),miR-182-Sp inhibitor (miR-182-5p inhibitor group),negative control mimic (NC mimic group) and negative control inhibitor (NC inhibitor group) were transfected respectively.The expression of miR-182-Sp was detected by reverse transcription-polymerase chain reaction (RT-PCR).The protein expression of FOXO3a was detected by Western blotting.The cell proliferation activity was detected by methyl thiazolyl tetrazolium (MTT) method.The cell apoptosis was detected by flow cytometry.The targeted relationship between miR-182-5p and FOXO3a was detected by dual-luciferase experiment.Results The miR-182-5p expression of A549 cells and BEASo2B cells respectively was 3.21 ±0.24 and 1.01 ±0.11,and the difference was statistically significant (t =14.209,P＜0.001).The miR-182-5p expression of NC mimic group,miR-182-5p mimic group,NC inhibitor group and miR-182-5p inhibitor group respectively was 1.09 ± 0.20,12.80 ± 1.10,1.03 ± 0.11and 0.47 ± 0.08,and the difference was statistically significant (F =87.872,P ＜ 0.001).The FOXO3a expression of the above four groups respectively was 118.34 ± 16.71,50.89 ± 11.58,125.33 ± 20.87 and 289.26 ± 34.51,and the difference was statistically significant (F =62.125,P ＜ 0.001).The 72 h proliferation activity of the four groups respectively was 1.12 ± 0.13,1.70 ± 0.14,1.07 ± 0.13 and 0.71 ± 0.11,and the difference was statistically significant (F =31.336,P ＜ 0.001).The proliferation activity of miR-182-5p mimic group was significantly higher than that of NC mimic group (P ＜ 0.05),and the proliferation activity of miR-182-5p inhibitor group was significantly lower than that of NC inhibitor group (P ＜0.05).The apoptosis rate of the four groups respectively was (5.51 t±1.80)％,(1.41 ±0.50)％,(6.24 ± 1.71)％ and (47.93 ± 5.12) ％,and the difference was statistically significant (F =211.081,P ＜ 0.001).The apoptosis rate of miR-182-5p mimic group was significantly lower than that of NC mimic group (P ＜ 0.05),and the apoptosis rate of miR-182-5p inhibitor group was significantly higher than that of NC inhibitor group (P ＜0.001).The miRNA target genes prediction software test results showed that miR-182-5p could act on FOXO3a 3' untranslated region (UTR).Compared with transfection NC mimic,co-transfection miR-182-5p mimic and FOXO3a-Wt could make luciferase activity of A549 significantly decreased (1.20 ±0.14 vs.0.62 ±0.10;t =5.839,P =0.004).Conclusion miR-182-5p can enhance proliferation and inhibit apoptosis of A549 cell by targeting FOXO3a.

Effect of Astragaloside Ⅳ on Regulation of PI3K/Akt/FoxO1 Signal in Kidney of Type 2 Diabetic Nephropathy Rats / 中国实验方剂学杂志

Objective:To study the protective effect of astragaloside (AS) Ⅳ on kidney in type 2 diabetic nephropathy rats and its regulation effect on phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O1(FoxO1) signaling, and investigate the mechanism of glycosides against type 2 diabetic nephropathy. Method:After 6 weeks of high-glucose and high-fat diet, rat models of type 2 diabetes were established by intraperitoneal injection of streptozotocin (STZ) (35 mg·kg-1) and randomly divided into model group, AS-Ⅳ (20, 40, 80 mg·kg-1) groups and metformin hydrochloride (positive) group. After 8 weeks of continuous administration, changes in body weight, kidney index, blood glucose, 24-hour urinary protein, urinary microalbumin, urinary creatinine, serum creatinine, and blood urea nitrogen were measured in each group; hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissues; Masson trichrome staining was used to observe the collagen expression level. Periodontium hexaammine silver (PASM) staining was used to observe the changes of basement membrane. Immunohistochemistry assay was used to detect phospho-FoxO1(p-FoxO1) protein expression, and Western blot was used to analyze the expression levels of autophagy marker protein PI3K, microtubule-associated protein 1 light chain 3 (LC3)/Ⅱ, B-cell lymphoma-2 (Bcl-2)/adenovirus E1B19 kDa interacting protein 3 (BNIP3), Beclin1, p-Akt, and Akt. Result:As compared with the normal group, the glomerular basement membrane of the model group was thicker; the extracellular matrix was increased; the mesangial was expanded; the collagen was significantly increased; the PI3K/Akt/FoxO1 signal was increased(PPPPPPConclusion:AS-Ⅳ may increase the autophagic activity of renal cells by inhibiting PI3K/Akt/FoxO1 signal, slowing down the development of type 2 diabetic nephropathy.

Effect of NMN on renal fibrosis of diabetic nephropathy rats and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the effect of niacinamide mononucleotide (NMN) on the fibrosis of renal cells in the rats with diabetic nephropathy (DN), and to elucidate the mechanism of NMN in regulating the fibrosis of renal parenchymal cells through silent information regulator 1 (Sirtl) and AKT pathways. Methods: The rat models of type 2 diabetes mellitus were induced by streptozotocin (STZ) and the model rats were randomly divided into experiment group (n= 30) and control group (n=10). The rats in experiment group were divided into diabetes + NMN group (n=15) and diabetes + PBS group (n=15). The rats in diabetes+ NMN group were given subcutaneous injection of NMN for 20 d and the rats in diabetes + PBS group were given 200 μL sterile PBS in the same way. Then the rats were decapitated and the kidney tissues were taken for section and protein extraction. The expression levels of Sirtl, AKT, p-Fox03a and Cav-1 proteins in kidney tissue of the rats in various groups were detected by Western blotting method and immuno-confocal focusing. The glomerular mesangial HBZY-1 cells were treated with high concentration of glucose (200 mmol · L-1) for 3-6 d, and then the cells were further randomly divided into 4 groups (treated with 0, 50, 100, and 200 mmol · L-1 NMN) and the cells only treated with 5. 6 mmol · L-1 glucose were regareded as control group. After 24 h culture, the cells were collected and the expression levels of Sirtl, AKT, and p-Fox03a proteins in the HBZY-1 cells in various groups were detected by Western blotting method. Results: Compared with diabetes +PBS group, the expression levels of Sirtl and AKT proteins in the renal parenchyma cells of the rats in diabetes+ NMN group were significantly increased (P<0. 01) and the expression levels of p-Fox03a and Cav-1 proteins in the renal parenchyma cells of the rats in diabetes + NMN group were also increased (P<0. 01). Compared with control group, the expression levels of Sirtl and AKT proteins in the HBZY-1 cells of the rats in 50 mmol · L-1 NMN group were significantly increased (P<0. 01), and the expression levels of Sirtl, AKT, and p-Fox03a proteins in the HBZY-1 cells in 100 and 200 mmol · L-1 NMN groups were increased significantly (P<0. 05 or P<0. 01). Conclusion: NMN can increase the expression levels of endogenous p-Fox03a and Cav-1 proteins in the glomerular cells of the DN rats by regulating the expression levels of Sirtl and AKT proteins, indicating that NMN and its analogues may play an important role in the prevention and treatment of the renal fibrosis of the DN rats.

Role of FOXO3a in hypoxia-induced cisplatin resistance in osteosarcoma U-2OS cells / 中国肿瘤生物治疗杂志

@# Objective:To investigate the role of FOXO3a in hypoxia-induced cisplatin (DDP) resistance in osteosarcoma cells. Methods: The FOXO3a expression was detected by RT-PCR and Western blotting in osteosarcoma U-2OS cell line under normoxia and hypoxia conditions. The effects of HIF-1α-siRNAand FOXO3a-siRNAon the expressions of HIF-1αand FOXO3a were detected by Western blotting. CCK-8 and Annexin V/PI assays were used to detect the function of FOXO3a in hypoxia-induced DDP resistance of U20S cells. Results: Hypoxia could significantly increase the mRNAand protein levels of FOXO3a in U-2OS cells (All P<0.05). The expression of FOXO3a was regulated by HIF-1α; compared with control group and HIF-1α-NC group, the FOX03a protein expression was significantly down-regulated in HIF-1α-siRNA group [(0.38±0.03) vs (0.89±0.08)， (0.91±0.07), all P<0.01]. Under hypoxia condition, FOXO3a-siRNA could decrease the tolerance of U-2OS cells to DDP [(38.50±2.83)% vs (61.75±5.73)%, P<0.01], and increase DDP-induced apoptosis of U-2OS cells [(73.41±6.13)% vs (32.38±2.23)%, (55.89±4.46)%,All P<0.05]. Conclusions: Hypoxia significantly enhanced DDP-resistance of U-2OS cells by increasing FOXO3a expression in a HIF-1-dependent manner.

Relationship between FoxO1 Expression and Wound Age during Skin Incised Wound Healing / 法医学杂志

Objective To investigate FoxO1 expression and its time-dependent changes during the skin incised wound healing. Methods After the establishment of the skin incised wound model in mice, the FoxO1 expression of skin in different time periods was detected by immunohistochemistry and Western blotting. Results Immunohistochemistry staining showed that FoxO1 was weakly expressed in a few fibroblasts of epidermis, hair follicles, sebaceous glands, vessel endothelium and dermis in the control group. The FoxO1 expression was enhanced in the epidermis and skin appendages around the wound during 6-12 h after injury, which could be detected in the infiltrating neutrophils and a small number of monocytes. FoxO1 was mainly expressed in monocytes during 1-3 d after injury, and in neovascular endothelial cells and fibroblasts during 5-10 d. On the 14th day after injury, the FoxO1 expression still could be detected in a few fibroblasts. The Western blotting results showed that the FoxO1 expression quantity of the tissue samples in injury group was higher than in control group. The FoxO1 expression peaked at 12 h and 7 d after injury. Conclusion FoxO1 is time-dependently expressed in skin wound healing, which can be a useful marker for wound age determination.

Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

BACKGROUND: Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. METHODS: PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. RESULTS: Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. CONCLUSION: Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.