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ObjectiveTo validate the alleviating effect of Gegen Qinliantang （GGQLT） on insulin resistance in db/db diabetic mice by regulating the silent information regulator 1 （SIRT1）/forkhead transcription factor O1 （FoxO1） autophagy pathway. MethodSeventy-five SPF-grade spontaneous type 2 diabetic db/db mice and 15 control db/m mice were selected and maintained on regular feed for one week before measuring blood glucose. They were randomly divided into six groups， with 15 mice in each group. The groups included a normal group （physiological saline， 0.2 g·kg-1）， a metformin group （0.2 g·kg-1）， high-， medium-， and low-dose GGQLT groups （31.9， 19.1， 6.9 g·kg-1）， and a model group （physiological saline， 0.2 g·kg-1）. They were orally treated with corresponding drugs for eight weeks， once daily. Fasting blood glucose （FBG） was measured using a Roche glucometer. Serum levels of high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， triglyceride （TG）， and total cholesterol （TC） were measured using an automated biochemical analyzer. Fasting serum insulin （INS） levels were determined using enzyme-linked immunosorbent assay （ELISA）， and the homeostasis model assessment of insulin resistance （HOMA-IR） was calculated. Western blot was used to detect the expression of Beclin-1， microtubule-associated protein 1 light chain 3 （LC3）， and SIRT1/FoxO1 autophagy pathway-related proteins in liver tissues. Immunohistochemistry was performed to assess the expression of SIRT1， FoxO1， Beclin-1， and LC3B proteins in liver tissues. Transmission electron microscopy was used to observe the formation of autophagosomes in the liver. ResultCompared with the normal group， the model group showed significant increases in FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.01）， and significant increases in the expression of SIRT1， Beclin-1， LC3， and FoxO1 proteins in liver tissues （P<0.01）. Transmission electron microscopy revealed the highest number of autophagosomes in the model group. Compared with the model group， the metformin group and the low-， medium-， and high-dose GGQLT groups showed significant decreases in serum FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.05， P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， LC3 （P<0.05， P<0.01）， and up-regulated FoxO1 protein （P<0.01）. Transmission electron microscopy showed a reduction in the degree of autophagy in the treatment groups. Compared with the metformin group， the medium- and high-dose GGQLT groups showed significant decreases in FBG， FINS， and TG levels （P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， and LC3 in liver tissues （P<0.05， P<0.01）， and reduced FoxO1 protein （P<0.01）. The high-dose GGQLT group showed reduced HOMA-IR， TC， LDL-C， and HDL-C levels （P<0.05， P<0.01）. Transmission electron microscopy revealed a significant reduction in autophagosomes in the medium- and high-dose GGQLT groups. ConclusionGGQLT can significantly improve glucose and lipid metabolism disorders， alleviate insulin resistance in db/db mice， and prevent and treat type 2 diabetes by activating the SIRT1/FoxO1 autophagy pathway.
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ObjectiveTo investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells. MethodA cell senescence model in the human ovarian granulosa-like tumor （KGN） cell line was induced by H2O2. A control group， a model group， and high-dose （1 μmol·L-1） and low-dose （0.5 μmol·L-1） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·L-1 H2O2 for 40 min. The effect of BBR on KGN cell proliferation was detected by cell counting kit-8 （CCK-8） assay. The effect of BBR on the senescence of KGN cells was detected by β-galactosidase staining. The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry. The effects of BBR on the mRNA expression of B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-3 （Caspase-3）， forkhead transcription factor O1 （FoxO1）， and catalase （CAT） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the effects of BBR on protein expression of silent information regulator1 （SIRT1）， superoxide dismutase 2 （SOD2）， c-Jun N-terminal kinase （JNK）， FoxO1， autophagy-associated protein microtubule-associated protein light chain 3Ⅱ （LC3BⅡ）， mammalian ortholog of yeast Atg6 （Beclin-1）， and ubiquitin-binding protein p62. ResultAfter H2O2 induction for 40 min， the cell proliferation rate of the model group decreased compared with that of the control group （P<0.01）， and the cell proliferation rates of the BBR groups increased compared with that of the model group （P<0.05）. The results of β-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group （P<0.01）， and the cellular senescence in the BBR groups was reduced compared with that of the model group （P<0.01）. As revealed by flow cytometry， compared with the control group， the model group showed increased apoptosis rate （P<0.01）， and compared with the model group， BBR groups showed decreased apoptosis rates （P<0.05）. Meanwhile， the ROS content in the model group increased compared with that in the control group （P<0.01）， and compared with the model group， the BBR groups showed reduced cellular ROS content （P<0.01）. The Real-time PCR results showed that compared with the control group， the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3 and FoxO1 （P<0.05）， and compared with the model group， the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax （P<0.05） and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells （P<0.05）. As revealed by Western blot results， SIRT1， SOD2， and p62 protein levels decreased in the model group compared with those in the control group （P<0.01）， and JNK FoxO1， LC3BⅡ， and Beclin-1 protein levels increased （P<0.05）. After BBR intervention， SIRT1， SOD2， and p62 protein levels increased （P<0.01）， and JNK， FoxO1， LC3BⅡ， and Beclin-1 protein levels decreased compared with those in the model group （P<0.05）. ConclusionBBR has an inhibitory effect on ovarian granulosa cell senescence， and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.
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Chronic kidney disease (CKD) has become a chronic disease that cannot be ignored all over the world. Renal fibrosis is the final result and key pathological features of CKD. The whole process of renal fibrosis is associated with the repair and healing of kidney damage, and this long-term chronic stimulation promotes the renal fibrosis to the kidney failure, it is characterized by continuous activation of myofibroblasts and excessive production and deposition of extracellular matrix. Renal fibrosis is a synergistic process mediated by a variety of signaling pathways, and TGF-β / Smad and Wnt / β-catenin signaling pathways are two of the most classic ones. FoxO1 is an important transcription factor in the human body, which is expressed in a variety of cell types and plays an important role in a variety of cell life activities. Current researches have shown that FoxO1 plays an important role in renal fibrosis. FoxO1 regulates the occurrence and development of renal fibrosis through various signaling pathways such as STAT, SIRT1, Wnt / β-catenin, PI-3K / Akt. For example, FoxO1 / STAT regulates TIF and renal tubule apoptosis, SIRT1 / FoxO1 regulates oxidative stress response and lipotoxicity, FoxO1 / β-catenin inhibits the Wnt / β-catenin signaling pathway. In addition, some related studies suggest that FoxO1 / Smad may play a role in renal fibrosis. The contribution and regulation of FoxO1 in the renal fibrosis are reviewed here in order to further clarify the pathogenesis of renal fibrosis, which may bring new clues and prospects for the treatment of CKD.
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OBJECTIVE: To investigate the effect of electroacupuncture (EA) on silent information regulator 1 (SIRT1), forkhead transcription factor O1 (FoxO1), and proopiomelanocortin (POMC) in the hypothalamus of rats with high-fat diet-induced obesity (DIO), as well as the mechanism of EA in regulating central appetite peptides to help lose weight. METHODS: Male Wistar rats were randomly divided into normal group, model group, EA group, EA＋inhibitor group, inhibitor group, and sham-operation group, with 10 rats in each group. High-fat diet was used to establish a rat model of DIO. The rats in the EA group and EA＋inhibitor group were given EA at "Fenglong" (ST40), "Zhongwan "(CV12)，"Guanyuan "(CV4), and"Zusanli" (ST36) with continuous wave at a frequency of 2 Hz and an intensity of 1 mA, for 10 minutes each time. The rats in the EA＋inhibitor group and inhibitor group were given tube placement in the third ventricle and injection of the specific SIRT1 antagonist EX-527. The rats in the sham-operation group were given tube placement in the third ventricle and injection of artificial cerebrospinal fluid. The above treatment was given 3 times a week for 8 weeks in total. Body weight, food intake, and Lee's index were observed before and after treatment. An automatic biochemical analyzer was used to measure the serum levels of total cholesterol (TC), triglyceride (TG), and free fatty acid (FFA), and Western blot was used to measure the protein expression of SIRT1, FoxO1, acetylated FoxO1(AC-FoxO1), and POMC in the hypothalamus. RESULTS: Before treatment, the model group, the EA group, the EA＋inhibitor group, the inhibitor group, and the sham-operation group had significantly higher body weight and food intake than the normal group (P<0.01), and the model group and the sham-operation group had a significantly higher Lee's index than the normal group (P<0.05). Compared with the model group after treatment, the EA group and the EA＋inhibitor group had significant reductions in body weight, food intake, TC, and the protein expression of AC-FoxO1 (P<0.01, P<0.05) and significant increases in the protein expression of SIRT1, FoxO1 and POMC (P<0.01, P<0.05); the EA group had significant reductions in Lee's index and the levels of TG, FFA(P<0.05，P<0.01)；the inhibitor group had significant increases in food intake, the serum levels of TC, TG, FFA(P<0.01) and significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01，P<0.05). Compared with the EA group, the EA＋inhibitor group and the inhibitor group had significant increases in body weight, food intake, Lee's index, the levels of TG, FFA and the protein expression of AC-FoxO1 (P<0.01, P<0.05) and significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01); the inhibitor group had significant increases in the serum levels of TC (P<0.01). Compared with the EA＋inhibitor group, the inhibitor group had significant increases in body weight, food intake, the serum levels of TC, TG, FFA, and the protein expression of AC-FoxO1 (P<0.01), as well as significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01). CONCLUSION: In rats with DIO, EA can effectively up-regulate the expression of SIRT1 in the hypothalamus, exert a deacetylation effect on FoxO1, and promote the expression of the downstream appetite-inhibiting peptide POMC, which may be one of the mechanisms of EA to help lose weight by regulating central appetite peptides in the obesity model.
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Objective To study the role and molecular mechanism of forkhead transcription factor O1 (FoxO1) on proliferation of mesangial cells( MCs) in diabetic rats. Methods Empty lentiviral vector( LV-pSC-GFP) and the constitutively active FoxO1 lentiviral vector(LV-CA-FoxO1) were constructed. Diabetic rat model was established and rats were divided into diabetes group(DM group), diabetes with LV-pSC-GFP group(NC group), and diabetes with LV-CA-FoxO1 group(CA group). The normal SD rats of the same age were considered as the normal control group(NG group). The lentiviral vector was injected into the renal cortex of diabetic rats in corresponding groups. Body weight, blood glucose, 24 h urinary protein, urine albumin, serum creatinine, and blood urea nitrogen was detected at the end of 2 weeks, 4 weeks, and 8 weeks. The ratio of kidney weight/ body weight was counted and the renal cortex was reserved for light microscopy, electron microscopy and frozen section after rats were sacrificed in different groups. The mRNA level of FoxO1 and p27Kip1 were detected by real-time PCR. The protein expressions of FoxO1, p-FoxO1, and p27Kip1 were tested by Western blotting. Results The renal pathological changes were obviously ameliorated in CA group. Compared with DM group, the mRNA and protein expression of FoxO1 and p27Kip1 were significantly increased in CA group (P 0. 05). The p-FoxO1 / FoxO1 ratio was decreased ( P 0. 05). Conclusion Overexpression of FoxO1 in kidneys of diabetic rats can inhibit the proliferation of mesangial cells, and may through up-regulating the expression of p27Kip1 delay the progression of diabetic nephropathy.
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Objective To study the effect and mechanism of forkhead transcriptionfactor O1( FoxO1) on proliferation of rat mesangial cells(MCs) cultured under high glucose conditions. Methods Constructing lentiviral vectors of LV-CA-FoxO1 and LV-siRNA-FoxO1 were used to upregulate or downregulate FoxO1. Moreover, negative control LV-NC-FoxO1 was also constructed. Rat MCs were separately cultured in normal glucose(5. 6 mmol/ L, NG group), only high glucose(30 mmol/ L, HG0 group), LV-NC-FoxO1 with HG(HG1 group),LV-CA-FoxO1 with HG (HG2 group), and LV-siRNA-FoxO1 with HG(HG3 group) for 72 h. MTT assay and flow cytometrywas were used to analyze cell proliferation and cell cycle distribution. The expression of FoxO1, cyclin-dependent kinase inhibitor (p27), cyclinD1, and cyclin-dependent kinase 4( CDK4) were detected by QRT-PCR and Western blot. Results The MCs proliferation rate in HG0 group was faster than that in NG group. Besides, there were no statistical differences in FoxO1 expression and proliferation rate of MCs between HG0 group and HG1 group. Nevertheless, LV-CA-FoxO1 promoted cell cycle arrest at the G1 phase and attenuated proliferation rate, along with upregulation of FoxO1 and p27 and downregulation of cyclin D1 and CDK4 in HG2 group ( all P < 0. 05). Moreover, degradation of FoxO1 by LV-siRNA-FoxO1 stimulated hyperproliferation of MCs, associating with decline of p27 and increase of cyclin D1 and CDK4 in HG3 group(all P<0. 05). Conclusion The proliferation of MCs induced by high glucose is regulated by utilizing transgenic technology targeted and regulated FoxO1 expression and consequently through FoxO1 / p27 signaling pathway. These findings indicate that FoxO1 seems to be a new therapeutic target for early diabetic nephropathy.
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To study the effects of forkhead transcription factor O1 (FoxO1) on the expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.Streptozotocin-induced diabetic rats were divided into three groups:diabetic rats (DM group),rats transfected with blank lentiviral vectors (diabetes mellitus plus LV-pSC-GFP group,LV-NC group),and rats which were transfected with lentiviral vectors carrying constitutively active FoxO1 (diabetes mellitus plus LV-FoxO1-AAA group,LV-CA group).Rats which received an injection of diluent buffer served as normal control.At 2,4,and 8 weeks after transfection,the levels of urine albumin,blood glucose,blood urea nitrogen,and serum creatinine were measured.Realtime PCR and Western blotting were performed to measure the mRNA and protein levels of FoxO1,COL4A3,COL4A5,and desmin in the renal cortex.Moreover,light microscope and electron microscope were used to observe the structural changes in glomerulus and podocytes.Compared with LV-NC and DM group,in LV-CA group,there was a significant increase in the mRNA and protein levels of FoxO1,and a distinct decrease in the levels of urine albumin,blood urea nitrogen and serum creatinine of rats (except at the twoweek time point) (all P＜0.05),the mRNA and protein levels of COL4A3,COL4A5,and desmin were all decreased (all P＜0.05),and pathological changes in kidney were also improved.Upregulating the expression of FoxO1 by transfecting with constructed lentiviral vectors can definitely improve the abnormal expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.
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Streptozotocin-induced diabetic rats were treated with resveratrol for 12 weeks.Compared with group of diabetic rats without treatment,the levels of urine albumin,serum creatinine,and blood urea nitrogen were significantly decreased and pathological changes in kidney were improved in treatment group ( P＜0.05 ).The mRNA expressions of FoxO1 and catalase were higher and FoxO1 phosphorylation level in diabetic rats treated with resveratrol was lower than that in diabetic rats without treatment (P＜0.05),suggesting that resveratrol may protect the kidneys of diabetic rats via regulating expression of FoxO1.
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Objective To study the effects and mechanisms of resveratrol (Resv) on the expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in rat mesangial cells (MCs) cultured in intermittent high glucose medium.Methods The MCs cultured in normal glucose ( 5.6 mmol/L) medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs ( FoxO1 shRNA ),and then cultured in intermittent high glucose medium (5.6mmol/L or 30 mmol/L,alternately every 8 hours ) and resveratrol ( Resv 20 μmol/L).The MCs cultured in normal glucose (5.6 mmol/L) medium served as normal glucose control group (NG),and the MCs exposed to iutermittent high glucose medium were divided into five groups:intermittent high glucose group (IHG),IHG+Resv group,IHG+FoxO1 shRNA group,IHG+ Resv + FoxO1 shRNA group,IHG + Negative control group ( shNC ),each group was cultured for 72 h.The level of reactive oxygen species (ROS) was assessed by Fluorescence microplate reader.The mRNA levels of Sirt1,Foxo1,AdipoR1,and AdipoR2 were assessed by RT-RCR.The protein levels of FoxO1,phosphorylation FoxO1 ( p-FoxO1 ),AdipoR1,and AdipoR2 were assessed by Western blotting.Results ( 1 )Compared with NG,intermittent high glucose significantly decreased the mRNA expression of Sirt1,but markedly increased the level of p-FoxO1.Furthermore,the mRNA and protein expression levels of AdipoR1 were obviously decreased while the level of ROS was enhanced in IHG ( all P＜0.05 ).(2) Compared with IHG,the mRNA and protein expression levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was enhanced in group IHG+FoxO1 shRNA ( all P＜0.05 ).( 3 ) Compared with IHG,after treating with Resv,the mRNA expression level of Sirtl was increased,whereas the level of p-FoxO1 was decreased.Moreover,the mRNA and protein levels of AdipoR1 was increased while the level of ROS was lowered in group IHG+Resv (all P＜0.05 ).(4) Compared with group IHG+Resv,the mRNA and protein levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was increased in group IHG+Resv+FoxO1 shRNA (all P＜O.05).(5) In addition,the mRNA and protein expression level of AdipoR2 showed no significant difference among these groups ( P＞0.05 ).Conclusion ( 1 ) Resveratrol significantly increases the expression of AdipoR1 in MCs induced by intermittent high glucose.( 2 ) FoxO1 plays an important role in regulating the expression of AdipoR1 by resveratrol.