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The development of anticancer drugs targeting AKT1 has been reported in a variety of cancers, but there are few related studies on Chinese medicinals targeting AKT1- In this study, Compound stomachache capsules (CSC) was used for inhibiting prostate cancer (PC) cells growth by targeting AKT1 in vitro and in vivo. Through mass spectrum, target prediction and bioinformatics analysis, it is found that 37 of CSC compounds have anticancer activity, and 6 compounds such as (+)-Magnoflorine, 7-hydroxycoumarin may be their main active components against prostate cancer- The results showed that CSC had significant in vitro inhibition on the growth of prostate cancer cells (P<0- 01), and the growth inhibition rate of PC3 cells reached about 35% at 80 μg/ mL- CSC also increased ROS production, and significantly promoted apoptosis (P <0- 01) and G
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BACKGROUND: The research on neovascularization in diabetic retinopathy is mostly limited to vascular endothelial growth factor, but 6-phosphofructoklnase-2/fructose-2,6-diphosphatase (PFKFB3) also plays a certain role. OBJECTIVE: To Investigate the effect of PFKFB3-SÍRNA on human umbilical vein endothelial cells (HUVECs) In high glucose environment. METHODS: HUVECs were divided Into four groups: Normal glucose control group (5.5 mmol/L glucose), normal glucose+PFKFB3-slRNA group (5.5 mmol/L glucose+PFKFB3-siRNA), high glucose group (30 mmol/L glucose), high glucose+PFKFB3-slRNA group (30mmol/L glucose+PFKFB3-slRNA). Western blot assay was used to detect the silencing effect of PFKFB3 expression. PFKFB3 with optimal silencing effect was selected for subsequent experiments. The tubule formation was detected by in vitro tubule formation assay. The expression of PFKFB3 mRNA was detected by real-time fluorescent quantitative PCR. The expression of PFKFB3 and AKT protein was detected by western blot. RESULTS AND CONCLUSION: PFKFB3-SÍRNA significantly inhibited the expression of PFKFB3 (P 0.05). Compared with the high glucose group, the tube formation ability of the high glucose+PFKFB3-siRNA group was significantly increased (P 0.05). Compared with the high glucose group, the ratio of p-AKT/AKT protein in the high glucose+PFKFB3-siRNA group was increased (P < 0.01). To conclude, siRNA silencing of PFKFB3 gene expression can inhibit the expression of PFKFB3 and improve tube formation in HUVECs. The mechanism may be related to the down-regulation of AKT expression.
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Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.
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Aim To investigate the effect of pyropheophorbide-a methyl ester-mediated photodynamic therapy (MPPa-PDT) on migration in human breast cancer MCF-7 cells as well as its possible molecular mechanism. Methods MCF-7 cells were randomly divided into control group, MPPa group, light group and MP- Pa-PDT group. Cells were co-cultured with MPPa at different concenlrations(0, 0. 5, 1,2,4 jimol • L"1) and exposed to light of different energy intensities, 0. 9, 1. 8, 2. 7, 3. 6 J/cm2). Cell viability was measured by CCK-8; production of ROS was detected by DCFH-DA staining; cell migration was mesured by Tr- answell migration assay and wound healing assay; the expressions of E-cadherin, PTEN, Akt, p-Akt were measured by Western blot. Results Compared with control group, MPPa group and light group, MPPa- PDT could significantly inhibit cell viability of MCF-7 cells in an energy-dependent manner. DCFH-DA staining suggested ROS in MPPa-PDT group was higher than that in the other three control groups; Transwell assay and wound healing assay showed cell migration in MPPa-PDT group was significantly inhibited; Western blot analysis indicated that the expression of E-eadhcr- in, PIXN in MPPa-PDT group increased, while p-Akt decreased. Conclusions MPPa-PDT inhibits MCF-7 cells' migration, and PTEN/AKT signal pathway may be involved in the process.
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Objective To discuss the anti-inflammatory effect and its possible mechanism of different insulin concentration on IκB, NF-κB and TNF-α mRNA in monocytes stimulated by lipopolysaccharide and to detect the key molecule P-Akt in PI3K/ Akt signaling pathway at the cellular level. Methods The peripheral blood (10 ml)is collected from 30 type 2 diabetics (T2DM). Then, the mononuclear cells are centrifugally separated based on density gradient and divided into five different groups:the control (Con)group; the low concentration insulin 100 mU/L(L-Ins)group; the combination of low concentration insulin 100 mU/L and LY-294002 10 μmol/L(L-Ins+LY) group; the high concentration insulin 1000 mU/L (H-Ins) group, The combination of high concentration insulin 1000 mU/L and LY- 294002 10 μmol/L(H-Ins+LY) group. Two subgroups were set in each category. After incubating for 24 hours, the lipopolysaccharide (100 ng/ml)was added and the mononuclear cell culture of peripheral blood was continued. One hour later, the activities of P-Akt, IκBα and NF-κB of one subgroup were detected using western blot; two hours later, monocytes of the other subgroup were collected and the level of TNF-a mRNA was detected using real-time PCR Results Compared with Con group, the expressions of P-Akt and IkB were higher in L-Ins group and H-Ins group (P<0. 05), the expressions of NF-κB and TNF-a mRNA were lower in L-Ins group and H-Ins group (P<0. 05). Compared with L-Ins group, the expressions of P-Akt and IκB were higher in H-Ins group (P<0. 05), but the expressions of NF-κB and TNF-a mRNA were lower (P< 0. 05). Compared with L-Ins + LY group (H-Ins + LY group), the expressions of IκB and P-Akt were higher(P<0. 05)and the expressions of NF-κB and TNF-a mRNA were lower in L-Ins group (H-Ins group)(P<0. 05). Compared with Con group, no significant variations were shown in the expressions of P-Akt, IkB and NF-κB in L-Ins+LY group and H-Ins+LY group (P>0. 05) except for the high expressions of TNF-a mRNA (P<0. 05). There is no significant difference between L-Ins+LY group and H-Ins+LY group (P>0. 05). Conclusion The direct anti-inflammatory effect of insulin is verified in a dose dependent manner. Insulin may regulate the synthesis and secretion of inflammatory cytokines by activating PI3K/Akt pathway, increasing IkB and affecting the state of NF-κB. Insulin may increase the synthesis and secretion of inflammatory cytokines through other pathways when the PI3K/Akt pathway is blocked.
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Objective To explore the expression of calreticulin (CRT) in gallbladder cancer tissue and its effect on the biological behavior in gallbladder cancer GBC-SD cells.Methods Immunohistochemistry and RT-qPCR were applied to detect the expression of CRT.Small interfering RNA was transfected into gallbladder cancer GBC-SD cells and Western blotting were used to detect the expression of CRT.The proliferation was determined by using cell counting kit-8 (CCK-8) and clone assays.Flow cytometry were applied to detect the apoptosis and cell cycle.Migration was detected by wound healing and transwell assays,respectively.The expression of p-Akt and MMP-9 were detected by using Western blotting.Results Expression of CRT in gallbladder cancer tissues is higher than adjacent cancer tissues and chronic cholecystitis tissues(t =5.571,P ＜ 0.05).The relative growth rate in the siCRT-1,siCRT-2 experimental group for 24 hours,48 hourrs were 71.5％ ±6.3％,79.5％ ±2.7％;62.6％ ± 8.8％,55.6％ ±2.6％,respectively.The apoptosis rate in the blank group,the negative control group,siCRT-1 and siCRT-2 group were 3.0％ ± 1.8％,4.7％ ± 1.3％,13.6％ ± 1.0％,20.0％ ± 4.0％,respectively.Wound healing assays showed that the wound closure ratio in the blank group,negative control group,siCRT-1 and siCRT-2 group were(0.67 ±0.02),(0.58 ±0.02),(0.22 ±0.01),(0.37 ±0.04),respectively.Transwell experiments showed that the numbers of migration of GBC-SD cells in the blank group,negative control group,siCRT-1 and siCRT-2 group were (302 ± 11),(297 ± 15),(178 ± 10),(165 ± 12),respectively,compared with the blank group and the negative control group,the relative growth rate for 24 hours and 48 hours was significantly lower,the apoptosis rate was higher,the numbers of migration was lower (F =29.310,118.618,69.651,144.515,190.145,P ＜ 0.05).Compared with the blank group and the negative control group,the expression of p-Akt and MMP-9 decreased after down-regulating the expression of CRT.Conclusions The expression of CRT in gallbladder cancer tissue was higher.CRT downregulation mediated changes of biological behaviors in gallbladder cancer may be associated with p-Akt/MMP-9 signal pathway.
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Objective Effect of fluoxetine on the expression of angiogenic factors in hippocampus of depressive rats.Methods 32 male SD rats (3 month) of SPF level were randomly divided into fluoxetine group (n=8),fluoxetine ± LY294002 intervention group (n=8),model group (n=8) and control group (n =8).Control group without any treatment was free to eat and water for 5 weeks.The rats in the other groups were modeled by chronic,mild and unpredictable multiphase stress.The fluoxetine group was treated with fluoxetine for 2 weeks after modeling,and the intervention group was given LY294002 intervention,then the fluoxetine treatment for 2 weeks,the model group was free to eat and drink for 2 weeks.At the end of the 5th week,all the rats were decapitated and the hippocampus was removed on the ice bath.Western blot was performed to detect the expression of angiogenic factors.Results Compared with the model group (vascular endothelial growth factor(VEGF) (0.44 ± 0.08),fetal liver kinase 1 (FLK1) (0.37 ± 0.15),protein kinase B1 (AKT1) (0.40±0.10),p-AKT1 (0.53±0.07)),the expression of VEGF (0.98± 0.13),FLK1 (1.09± 0.21),AKT1 (1.05±0.05) and p-AKT1 (1.01±0.13)in hippocampus of fluoxetine group were significantly increased,and the differences were statistically significant (P＜ 0.01).And the expression of VEGT,FLK1,p-AKT1 in fluoxetine group increased compared with those of fluoxetine ± LY294002 intervention group (VEGF (0.55±0.08),FLK1 (0.55±0.14) and p-AKT1 (0.60±0.05)),and the difference were statistically significant (P＜0.01).Conclusion Fluoxetine can increase the expression of VEGF/FLK1 and p-AKT1 proteins in the hippocampus of depressed rats,which in turn may be involved in the regeneration of hippocampal blood vessels.This effect of fluoxetine may be related to the PI3K/AKT signaling pathway.
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Aim To investigate the protective mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury in rats.Methods In vivo: the cerebral ischemia-reperfusion injury model was established by middle cerebral artery occlusion(MCAO)via suture method in rats;the rats were injected anisodine hydrobromide(1.2,0.6,0.3,0.15 mg·kg-1);the morphological changes were detected by HE staining;the Nissl staining was used to count the number of surviving neurons;the activity of CAT and LDH,the LPO contents in the brain tissue were measured;the expressions of Bax,Bcl-2,caspase-3 and p-Akt in brain tissue were detected by Western blot.In vitro: Western blot assay was used to determine the expression of Bax,Bcl-2,caspase-3 and p-Akt protein expression in the OGD-R model of PC12 cells.The signal pathway of anisodine hydrobromide was identified.Results Anisodine hydrobromide with the dose of 0.15 mg·kg-1 could significantly lessen the morphological changes,and improve the number of surviving neurons;the dose of 0.3 and 0.15 mg·kg-1 could significantly improve the activity of CAT;the dose of 0.3 mg·kg-1 could significantly reduce the contents of LPO in the rat brain tissue;the dose of 1.2 mg·kg-1 could significantly decrease the activity of LDH;the dose of 0.15～1.2 mg·kg-1 could inhibit the expression of Bax,promote the expression of p-Akt in rat brain tissue.All the doses except 0.15 mg·kg-1 could promote the expression of Bcl-2 in rat brain tissue.In vitro,the results showed that anisodine hydrobromide in 25～100 μmol·L-1 could significantly improve the expression of Bcl-2 and the ratio of Bcl-2/Bax,and the dose of 50 μmol·L-1 could significantly improve the ratio of p-Akt/Akt.Conclusion The mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury model rats might be related to its anti-oxidative activity and the activation of Akt.
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AIM:To study the effect of diallyl trisulfide (DATS) on experimental corneal neovascularization (CNV) in rats induced by corneal suture and detect the expression of vascular endothelial growth factor (VEGF) and p-AKT in rats cornea.METHODS:The rat model of corneal neovascularization (CNV) was induced by corneal suture.Rats were randomly divided into Group A:physiological saline control group containing DMSO (10 rats);Group B:25μmol/L DATS treatment group (10 rats);Group C:50μmol/L DATS treatment group (10 rats);Group D:100μmol/L DATS treatment group (10 rats);Group E:200μmol/L DATS treatment group (10 rats).The occurrence and development of CNV were observed by slit-lamp microscope at 7d after suture,and the area of CNV were calculated.Two weeks later,HE staining was used to observe the pathological organization form of each cornea,and RT-PCR and Western blot were used to detect the expression of VEGF mRNA and protein expression of VEGF and p-AKT between each groups.RESULTS:The blood vessel area of Group C,D and E was compared with that of Group A,the difference was statistically significant (P＜0.05);HE slice showed corneal edema,angiogenesis and inflammation infiltration situation gradually reduced comparing with the Group A,with the increase of concentration of DATS.RT-PCR showed the expression of VEGF mRNA in Group B,C,D,and E decreased compared with the Group A,and the difference was statistically significant (P＜0.05).Western-blot showed that the expressions of VEGF and p-AKT in Group B,C,D and E decreased gradually compared with those in Group A,and the difference was statistically significant (P＜0.05).CONCLUSION:DATS can inhibit corneal neovascularization of the rats induced by suture.Its mechanism may be associated with suppression of VEGF secretion,down-regulation of VEGF and inactivation of p-AKT.
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Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K⁺ balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-Akt, ERK, p-ERK protein in both normal kidney and K⁺-deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-Akt was increased gradually in K⁺-depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-ERK was markedly increased in K⁺-depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K⁺-depleted diet 2 weeks. The localization of p-Akt proteins in K⁺-depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K⁺-depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K⁺-depleted diet 2 and 3 weeks. The localization of p-ERK proteins in K⁺-depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K⁺-depleted diet 2 weeks. Taken together, we suggest that the expression of p-Akt was gradually increased in K⁺-depleted groups of kidney, but the expression of p-ERK was markedly increased in K⁺-depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction.
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Animals , Rats , Alkalosis , Blotting, Western , Diet , Extremities , Hypokalemia , Ion Channels , Ion Transport , Kidney , NF-E2-Related Factor 2 , Phosphorylation , Phosphotransferases , Signal Transduction , Transcriptional ActivationABSTRACT
Objective To determine the expression of Caspase 8 and phospho-Akt(p-Akt)in condyloma acuminatum(CA)lesions, and to evaluate their significance. Methods Skin lesion samples were collected from 30 patients with CA, cancer tissue samples from 20 with cervical cancer, and normal skin samples from 20 healthy controls. All the samples were subjected to paraffin embedding. An immunohistochemical study was conducted to determine the expression and distribution of Caspase 8 and p-Akt in the above samples. Results The expression rate of Caspase 8 was significantly lower in CA lesions (23.33%)than in normal skin samples(90%, P < 0.01)and cervical cancer lesions(80%, P < 0.001). Moreover, the expression rate of p-Akt in CA lesions(93.33%)was significantly higher than that in the normal skin samples(90%, P<0.001), but lower than that in the cervical cancer lesions(95%, P<0.001). No significant correlations were observed between the expression of Caspase 8 and p-Akt in either CA lesions or normal skin samples. However, the expression of Caspase 8 was positively correlated with the expression of p-Akt in cervical cancer lesions(r=0.369, P<0.05). Conclusion Both suppressed apoptosis initiation of Caspase 8 and anti-apoptotic effect of p-Akt may be involved in the occurrence and development of CA.
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Objective To study the relationship between the expression of p-AKT and HIF-1αprotein and the response of breast cancer to neoadjuvant chemotherapy.Methods A retrospective study was performed on 70 cases of breast cancer patients receiving 4-6 cycles of TEC neoadjuvant chemotherapy in Wenzhou people's hospital from January 2014 to December 2016.Immunohistochemistry was applied to the detection of p-AKT and HIF-1αexpression before chemotherapy.Neoadjuvant chemotherapy response was evaluated according to the postoperative pathology.Pathological response to G4 or complete response were considered to be response efficaciously.Chi-square test and Fisher's test were applied to analyze the correlation between these index and the efficacy of neoadjuvant chemotherapy.Results The response rate of neoadjuvant chemotherapy was 52.9%(37/70)among the 70 cases, among which the pathological complete response(PCR)was 21.4%(15/70).The positive expression of p-AKT and HIF-1α were 64.3%(45/70)and 61.4%(43/70)respectively.Statistical analysis shows that p-AKT and HIF-1α expression were associated with the response of neoadjuvant chemotherapy of breast cancer(P=0.002, P=0.035), and also associated with the PCR(P=0.001, P=0.015).Conclusion p-AKT and HIF-1αexpression in breast cancer reduce the sensitivity of neoadjuvant chemotherapy to breast cancer.Patient with negative expression may benefit more from neoadjuvant chemotherapy.
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Objective To observe the effects of cyclosporin A on the expression of phosphorylated AKT in hepatocytes, and to investigate the mechanism of insulin resistance caused by cyclosporin A. Methods This study included two parts. 1. In vitro experiment:Human hepatocyte HL77022 cell line was cultured at different concentrations of cyclosporin A (0?1 μmol/L, 1 μmol/L, 5 μmol/L) for 48 hours. The expressions of phosphorylated AKT ( P?AKT) in HL77022 cells were measured by Western blot assay. 2. Rat in vivo experiment: SD rats were randomly divided into 2 groups. The rats in the control group were administrated with distilled water 1 mL/Kg/d. The rats in the cyclosporine group were administrated with cyclosporine 25 mg/Kg/d. The total experiment time was 5 months. The levels of fasting blood glucose and insulin were tested at the end of the experiment. The insulin resistance index and insulin sensitivity index were calculated. The expression of P?AKT in the rat hepatocytes was measured by immunohistochemistry. Results 1. Each group of the HL7702 cells treated by CsA ( 0?1 μmol/L, 1 μmol/L, 5 μmol/L ) showed a significantly decreased expression of P?AKT (P<0?05, P<0?01, and P<0?01). 2. After 5 months of therapy, the fasting blood glucose level of rats in the cyclosporine group was 9?28 mmol/L, indicating that cyclosporine?induced diabetic rat models were established. The insulin sensitivity index in the cyclosporine group was lower than that in the control group ( P<0?05 ) . The expression of P?AKT in liver in the cyclosporine group was significantly lower than that in the control group ( P <0?05) . Conclusions Therapeutic dose of cyclosporine has hyperglycemic effects on rats. Cyclosporine can reduce the expression of phosphorylated AKT in hepatic tissue in rats and also decrease the expression of P?AKT in human hepatocyte HL77022 cells, which indicate that cyclosporine may cause insulin resistance by interfering PI3K/AKT signal transduction pathway.
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Objective To observe the effects of tacrolimus on blood glucose, insulin, expressions of protein phos-phatase 2A and P-AKT in rats in order to explore the mechanism of hyperglycemic action of tacrolimus. Methods Sixty male SD rats (body weight 89. 83 ±4. 44 g) were randomly divided into tacrolimus group (n =40) and control group (n=20). The rats in the tacrolimus group were administrated with tacrolimus 4 mg/kg daily. The rats in the control group were given the same amount of normal drinking water daily. The rat body weight, fasting blood glucose concentration and blood concentration of tacrolimus were measured monthly. All rats were killed at 5 months after the tacrolimus administra-tion. The serum insulin levels were detected by radioimmunoassay method. The expressions of PP2A and P-AKT in liver tissues were assessed by immunohistochemistry. Results After two months of administration, the blood glucose levels in the tacrolimus group were significantly higher than those in the control group. The HOMA-IR in tacrolimus group was signif- icantly higher than that in the control group P<0. 05 ) . ISI was significantly lower than that in the control group ( P<0. 05). Immunohistochemical examination showed that the expression of PP2A in hepatocytes in the tacrolimus group was increased compared with the control group, while expression of P-AKT in hepatocytes of the tacrolimus group was decreased than that in the control group. Conclusions Tacrolimus can induce necrosis of islet cells, decrease of the amount of islet cells and insulin secretion, decease of sensitivity to insulin, and increase the resistance to insulin, therefore, leading to in-crease the blood glucose level in rats. The expression of PP2A in hepatocytes in the tacrolimus group is increased, while the expression of P-AKT is decreased. Interfering of insulin signal transduction pathways may be involved in the hyperglyce-mic effects of tacrolimus.
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OBJECTIVE: To screen the best combination of extractum of Robinia-living trametes and chemotherapy and investigate the action mechanism of Robinia-living trametes against the apoptosis of human gastric cancer cell MGC803. METHODS: MGC803 Cells were treated with different concentrations of Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in vitro. The inhibitory rate of cells was measured by MTT assay. Morphological changes were observed with inverted microscope. The apoptosis rate of MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by FCM. The protein expression of P53 and p-Akt in MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by Western blot. RESULTS: The viability of MGC803 cells was reduced by Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in a concentration- and time-dependent manner (P<0.01). Under reverse microscopy, cell body shrinking, nuclear pyrosis, and nuclear fragmentation were observed. The higher concentration, the longer treatment time, the more cells died. Compared with monotherapy, the combination of Robinia-living trametes and chemotherapy could reduce the survival rate of MGC803 cells. The protein expressions of P53 in MGC803 cells treated with combination of drugs was up-regulated, while that of P-Akt was down-regulated. CONCLUSION: The apoptosis of MGC803 cells in vitro may be induced by the inhibitory effect of the combination of Robinia-living trametes and 5-Fu on PI3K/Akt signaling pathway. Combination therapy of Robinia-living trametes and 5-Fu is potentially more effective in inhibition of tumor cells than monotherapy of Robinia-living trametes.
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Aim To investigate the effect of simvastatin ( Sim ) on endogenous antioxidant system after acute myocardial infarction ( AMI ) and its potential mecha-nisms. Methods The acute myocardial infarction ( AMI ) rat models were made by ligation left anterior descending of coronary artery. Then the successful models were randomly divided into myocardial infarc-tion group ( MI group) and simvastatin group ( Sim,20 mg·kg-1·d-1), another group without ligation left anterior descending of coronary artery served as sham group(Sham group). The Sim group was administered simvastatin by gavage for 7 days. MI group and Sham group received saline. Hemodynamic parameters, lipid levels, troponinI ( c-TnI ) and lactate dehydrogenase ( LDH) concentrations were examined after 7days, and the levels of superoxide dismutase ( SOD) and glutathi-one peroxidase ( GP) of myocardial antioxidant system were detected by ELISA. The expression of cardiac p-Akt and p-eNOS protein were detected by Western blot. Results Acute myocardial infarction significant-ly lowered cardiac hemodynamic parameters, increased serum c-TnI and LDH levels, lowered levels of SOD and GP, and lowered the expression of p-Akt and p-eNOS protein. However, Sim could effectively prevent the deterioration of cardiac function, reduce serum c-TnI and LDH levels, increase levels of SOD and GP, and increase p-Akt and p-eNOS protein expression. Conclusion Early using Sim can effectively improve heart function after acute myocardial infarction, acti-vate myocardial antioxidant system,and reduce myocar-dial necrosis, which may be related to increasing the expression of p-Akt and p-eNOS.
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This study was aimed to explore the mechanism of baicalin on high altitude cerebral hypoxia-ischemia on mice and its influence on related target protein expressions. Morris water maze was used to screen 50 Kunming mice, which were randomly divided into the model group, control group, the low dose (0.05 mg·kg-1), middle dose (0.20 mg·kg-1) and high dose (0.60 mg·kg-1) baicalin group, with 10 rats in each group. The space memory and learning ability of mice were tested. The animal cabin with low oxygen (simulating at 4 000 m altitude) was used to establish the stable high altitude cerebral hypoxia-ischemia mouse model. Changes on SOD content, GSH-PX activities and MDA content in hippocampal tissues of mice were detected. The expressions of different target proteins, including cleaved-caspase 3, P-AKT, GFAP, Bax and Bcl-2 in brain stem of mice were detected by western blot. The results showed that the latent period of the model group was obviously longer than that of the control group (P < 0.05). The latent period of high dose baicalin group was shorter than the model group with significant difference (P< 0.05). Therefore, the best effective dose of baicalin was 0.60 mg·kg-1. Compared with the control group, the content of MDA in the hippocampal tissues of mice in the model group was significantly increased; the SOD and GSH-PX activity were obviously reduced (P < 0.05). Compared with the model group, the SOD and GSH-PX activity were obviously increased in the brain tissues of mice in the high dose baicalin group; and the content of MDA was obviously reduced (P < 0.05). From the level of protein changes, the stripes of cleaved-caspase 3, P-AKT, GFAP protein expressions in the model group were strengthened compared to the control group; the ratio of Bax/Bcl-2 was also obviously increased (P < 0.05). The expression of the baicalin group was lower than that of the model group (P < 0.05). Among them, the expression of the high dose baicalin group was the lowest. It had certain dose-response relationship. It was concluded that baicalin had protective effect on high altitude cerebral hypoxia-ischemia. Its mechanism may be related to its powerful oxidation resistance and its inhibition on expression of different target proteins, including cleaved-caspase 3, P-AKT, GFAP, Bax, Bcl-2 for the change of apoptotic pathway.
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Aim To investigate the effects of cell pro-liferation and activation in HSC-T6 cells by inhibiting the expression of EZH2 , and its partial relevant mech-anism. Methods By introducing the inhibitor DZNep in activated HSC-T6 cells stimulated by TGF-β1 , the protein expression levels of EZH2, p-ERK, p-AKT andα-SMA were detected by Western blot. The siRNA targeting EZH2 was designed and synthesized according to its nucleotide sequence, and their corresponding ex-pression vectors were constructed and transfected into HSC-T6 cells with LipofectamineTM 2000. The prolifer-ation of HSC-T6 cells was determined by MTT. And the protein expression levels of EZH2, p-ERK, p-AKT and α-SMA were measured by Western blot. Results By introducing the inhibitor DZNep in activated HSC-T6 cells stimulated by TGF-β1 , it effectively de-creased the protein levels of EZH2 and also the protein levels of p-ERK, p-AKT and α-SMA. By introducing EZH2-siRNA in activated HSC-T6 cells, it effectively inhibited the cell proliferation, and also the protein levels of EZH2, p-ERK, p-AKT andα-SMA. Conclu-sion Silencing EZH2 expression inhibits HSC-T6 cell proliferation and activation, and EZH2 may be a poten-tial therapeutic target gene for hepatic fibrosis.
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Objective To investigate the effects of isoliquiritigenin on the invasive ability of human gastric carcinoma SGC7901 cells, and its molecular mechanisms thereof. Methods The logarithmic phase human gastric carcinoma SGC7901 cells were divided into control group (normal cell culture fluid) and isoliquiritigenin group (isoliquiritigenin solu?ble in cell culture fluid, the concentrations were 10, 25, 50 and 100 μmol/L respectively). Each group had four repeated holes. The proliferation of SGC7901 cells were detected with MTT assay after 24 h, 48 h and 72 h of culture. The experimen?tal drug concentration and action time were researched for the subsequent experiments. The in vitro invasion abilities of SGC7901 cells were assessed with Transwell test. The expression levels of MMP9, Akt and P-Akt were detected by Western blot assay. Results The proliferation of SGC7901 cells were inhibited by 10μmol/L isoliquiritigenin, which can be signifi?cantly inhibited by 25, 50 and 100μmol/L isoliquiritigenin in a concentration-dependent and time-dependent manner. The half inhibitory concentrations (IC50) of 24, 48 and 72 h were 52.48, 44.49 and 32.50μmol/L, respectively. Therefore, the 25, 50 and 100μmol/L isoliquiritigenin were selected as the subsequent experimental drug concentration, and 24 h was used as the action time. Compared with the control group (209.75±9.29), the membrane cell number of 25μmol/L (138.50±10.15), 50μmol/L (89.50 ± 16.56) and 100μmol/L (45.00 ± 8.08) decreased gradually (F=267.948,P<0.05). There was no signifi?cant difference in the expression level of Akt protein between four groups (F=1.492). The expression levels of P-Akt and MMP9 were gradually decreased with the increase of the isoliquirigenin concentration (F=359.219 and 431.324,P<0.05). Conclusion Isoliquiritigenin can obviously inhibit invasion ability of SGC7901 cells, which may be related to the down reg?ulation of the signal transduction pathway protein PI3K/Akt and the down steam protein MMP9.
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Objective To explore the effect of calreticulin (CRT) on cell proliferation and invasion of human hepatocellular carcinoma cell line SMMC-7721 and HepG2.Methods SMMC-7721 and HepG2 cells were transfected with small interfering RNA (siRNA).The transfection rate was detected by immunoflurescence and western blot.The cell proliferation,invasion and apoptosis of SMMC-7721 and HepG2 cells were determined by using cell counting kit-8 (CCK-8) assays,transwell assays and flow cytometry,respectively.The p-Akt and Akt levels were detected by western blot.Results The growth inhibition rate in the siRNA experimental group of SMMC-7721 and HepG2 cells for 24,36 and 48 h were (41.0 ±2.2) ％,(46.5 ±1.6)％,(59.7 ±2.2)％ and (36.8 ±2.7)％,(47.3 ± 1.8)％,(61.5 ±3.2)％,respectively.The apoptosis rate after down-regulating the expression of CRT in SMMC-7721 and HepG2 cells for 36h were (45.2 ± 9.1) ％ and (48.9 ± 8.0) ％,respectively.Compared with the blank group and the negative control group,the growth inhibition rate in the siRNA experimental group was lower (P ＜0.05),but the apoptosis rate was significantly higher (P ＜ 0.05).Transwell experiments confirmed that the numbers of invaded SMMC-7721 and HepG2 cells in the blank group and the negative control group and siRNA experimental group were (96.8±7.3),(95.6±5.4),(34.0±4.2) and (124.0 ±9.9),(121.6 ±7.0),(70.4±9.5),respectively,indicating that cell invasion in the siRNA experimental group was significantly suppressed (P ＜ 0.05).The expression of p-Akt was decreased (P ＜ 0.05) after down-regulating the expression of CRT for 36h.Conclusion CRT gene silencing by siRNA can inhibit the SMMC-7721 and HepG2 cell proliferation and invasion,but increase the cell apoptosis by regulating PI3K/Akt signal pathway.