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ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions. ResultAs compared with the control group， the glucose consumption in the model group was significantly decreased （P<0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （P< 0.01）. As compared with the control group， the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （P<0.05， P<0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （P<0.05， P<0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （P<0.01）， and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （P<0.05， P<0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （P<0.05， P<0.01）. ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.
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ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions. ResultAs compared with the control group， the glucose consumption in the model group was significantly decreased （P<0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （P< 0.01）. As compared with the control group， the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （P<0.05， P<0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （P<0.05， P<0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （P<0.01）， and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （P<0.05， P<0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （P<0.05， P<0.01）. ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.
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It is the need of the day to identify the new anticancer herbal drug, which not only in possession of good anticancer effects but also cost effective. Here we are presenting such an anticancer Ayurvedic herb which is used since the centuries for the treatment of different diseases of diverse origin. Eclipta alba Hassk., also called as Bhringraj is very important medicinal herb in many medicinal formulations. Though it is commonly used for hair growth, many evidences found its hepatoprotective activity. Here we are presenting all aspects about Bhringraj in terms of qualitative and quantitative values and we have also tried to prove the anticancer activity of it for hepatic cancer. We have used the aqueous extract of Eclipta alba Hassk. for phytochemical analysis, TLC, HPLC analysis to test active chemical components in it. Extract showed presence of many active chemical components which were responsible for its anticancer activity. In vitro study we used the aqueous extract of Eclipta alba Hassk. for the evaluation of its effects on HepG2 (Human liver cancer cell line). The SRB assay results were used to evaluate the anti-cancer activity of the extract. The effects of whole plant extract on cancer cell line were studied. Percentage of cell growth and cell viability were calculated from tabulated result values of srb assay. The experiment revealed that the average percentage of growth inhibition was 68.74%. Cell viability SRB assay also showed significant growth inhibition, at the same time statistical analysis of SRB assay also proved significant results. The research performed here is very useful for set up of different extract studies of Bhringraj for its anticancer activity.
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Objective To evaluate the effect of Ji Tang Zhi on glucose metabolism in insulin resistance (IR) HepG 2 cell line,and to explore the related mechanism.Methods The HepG2 cells were incubated in culture medium addition of 10-7 mol/L insulin for 24 h to establish the IR cell model.Effect of Ji Tang Zhi on rate of glucose absorption in HepG2 cell was detected by the method of glucose oxidase-peroxidase (GOD-POD).We performed an MTT assay to determine cytotoxicity effects of Ji Tang Zhi on HepG2 cell line.The expression of p-IRS-1 Ser307,PI3K and GLUT-4 were detected by Western blotting.Results Incubated with 10-7 mol/L insulin for 24 h,the insulin resistance cell model had been built.Compared with model group,the rate of glucose absorption of cell treated with JTZ (30 ～ 120 μg/mL) was significantly improved.According to model cells,the expression of GLUT-4 and PI3K decreased significantly compared to control cells.While the expression of p-IRS-1 Ser 307 was inhibited and GLUT-4 and PI3K expression were increased in IR cells after treated with JTZ (30 ～ 120 μtg/mL).Conclusion JTZ exert beneficial effects on hyperglycosemia in IR cell line possibly through regulating the levels of GLUT-4,p-IRS-1 Ser307 and PI3K in HepG2 cell.
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Objective:To investigate the effects of ursolic acid on proliferation and apoptosis of hepatic carcinoma cell line HepG2 and its partial mechanism.Methods:The human HepG2 cells were cultured by different concentrations of ursolic acid.The inhibitions of ursolic acid on cell proliferation were determined by using MTT.The effects of ursolic acid on on cell apoptosis and cell cycle were detected by using flow cytometry.The expressions of pERK1/2 and Cyclin D1 proteins after culturing by different concentrations of ursolic acid were tested by using Western blotting.Results:The human hepatoma HepG2 cell proliferations were inhibited by different concentrations of ursolic acid,and the effects showed dose and time-dependent manner(P＜0.05).The cell apoptosis rate for human hepatoma cell line HepG2 was achieved to the maximum under 60 μ mol/L ursolic acid for 72 h,which was (78.723 ± 3.623)％.Ursolic acid could significantly increase the Go/G1 phase cells proportion,and induce HepG2 apoptosis.Ursolic acid could inhibit the expressions of pERK1/2 and Cyclin D1 proteins,and inhibitory effects showed more apparent along with the concentration and time gradually increasing.Conclusion:Ursolic acid could inhibit the proliferation of human hepatic carcinoma HepG2 cell line,block of cells in G0/G1 phase,and promote the cell apoptosis.This might be related with down-regulation of the expressions of pERK1/2 and Cyclin D1 proteins.
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OBJECTIVE: To study the effect of a novel naphthalimide-polyamine conjugate on apoptosis of human hepatocellular carcinoma HepG2 cell line. METHODS: MTT assay was used to evaluate the cell inhibition rate. The cellular morphous was detected with AO/EB/Hoechst staining, the ROS was detected with DCFH-DA staining, the mitochondrial membrane potential (ΔΨm) was detected with Rh123 staining, the expression of caspase-9 and PARP-1 was detected by immunofluorescence method. The cell cycle was detected by HCS. Apoptosis of HepG2 cells was quantified by flow cytometry using Annexin V/PI stain. RESULTS: Proliferation of HepG2 cells was inhibited significantly by the naphthalimide-polyamine conjugate in a dosage dependant manner. Under HCS, some HepG2 cells underwent a typical apoptotic morphologic change and the expression of ROS and caspase-9 was increased. The expression of PARP-1 and the mitochondrial membrane potential (ΔΨm) was detected decreased. Flow cytometry indicates 64.12% HepG2 cells were induced apoptosis after 48 h incubation with 25 μmol·L-1 naphthalimide-polyamine conjugate. CONCLUSION: The naphthalimide-polyamine conjugate could significantly induce the apoptosis of HepG2 cells by the mitochondrial pathway. The mechanism is concerned with increasing ROS, decreasing the mitochondrial membrane potential, upgrading caspase-9 and decreasing the expression of PARP-1.
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@#This study was aimed at isolating a peptide with hypoglycemic activity from dried red deer antler. A peptide from dried red deer antler of Tarim in Xingjiang was extracted with acetate buffer(pH 3. 5), followed by precipitation with ethanol, ultrafiltration and separation via ion exchange chromatography, gel filtration and reversed phase chromatography. The molecular mass of the peptide was detected via MALDI TOF/MS and the peptide was identified by LC-MS/MS. The activities of the peptide to promote islet β cells proliferation and recovery the damage induced by streptozotocin(STZ)were detected by MTT, and the effect of the peptide on glucose consumption of hepatocyte was determined. The peptide, named as CAP, was obtained from dried red deer antler through purification with the molecular mass of 6 804 via MALDI TOF/MS. The result of LC-MS/MS and the comparison in relative database showed that CAP was a novel peptide. CAP stimulated the proliferation of pancreatic cells and recovery of STZ-damaged pancreatic cells, as well as increased glucose consumption of HepG2.
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ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose) polymerase (PARP-1) inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P＜0.01). Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.
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A series of new thiopyrimidine derivatives were synthesized via the reaction of ethyl cyanoacetate with thiourea and the appropriate aldehydes namely, 3-methoxy-benzaldehyde, 2, 5-dimethoxy-benzaldehyde and 3,5- dimethoxy-benzaldehyde to give the corresponding pyridine thiones 1a-c. Compounds 1a-c were then chlorinated to give the corresponding chloro compounds 2a-c, which then underwent variant cyclocondensation reactions to afford different cyclized compounds 3-10. On the other hand, 1a-c were condensed with monochloroacetic acid and different aldehydes to give 11-14. Some of the new derivatives were selected for cytotoxicity evaluation against HepG2 cell line in comparison to 5-FU as a reference drug. Among all tested compounds, compound 4a was the most potent with IC50 value of 13.18 μM. Furthermore, a docking study of the most active compounds was carried out with thymidylate synthase enzyme. Structures of all new compounds were elucidated by their correct elemental analysis and spectral data.
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Objective: To observe the effect of bevacizumab on the proliferation of human heptoma cell line HepG2. Methods: The expression of vascular endothelial growth factor(VEGF) and its receptors (VEGFRs) in HepG2 cells were examined by immunocytochemical staining and RT-PCR; ELISA was used to determine the level of VEGF in culture supernatants of HepG2 cells. The proliferation of HepG2 cells was analyzed by MTT assay after treatment with rh VEGF and bevacizumab separately; the expression of VEGF was examined by RT-PCR and Western blotting. Results: VEGF and VEGFRs (Flt-1 and KDR) were expressed in human HepG2 cells. rhVEGF increased the proliferation of HepG2 cells in a dose-dependent manner within a concentration range of 0-100 ng/ml; bevacizumab inhibited the proliferation of HepG2 cells; the inhibition rates were (8.76% ± 1.15)%, (26.83 ± 1.20)%, (31.87 ± 1.30)% and (28.20 ± 1.28)%, when the concentrations of bevacizumab were 0. 1, 1, 10,and 20 μg/ml,respectively. Expression of VEGF in the HepG2 cells was increased by rhVEGF and inhibited by bevacizumab. Conclusion: Bevacizumab might inhibit the proliferation of HepG2 cells through blocking the effect of VEGF.
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Insulin resistance in insulin sensitive organ results in metabolic disorder such as hyperglycemia, hyperinsulinemia and hyper triglyceridemia which are common features of type 2 diabetes.Insulin resistance in liver cells mainly causes impaired glycogen synthesis, failed to suppress glucose production which is the major contribution to hyperglycemia.FGF-21 as a new metabolic regulator can control fasting blood glucose.The mechanism of FGF-21 effects on regulating plasma glucose has little to known.In order to establish an in vitro insulin resistant model of liver cells and evaluate the effects and mechanism of FGF-21 on glucose metabolism in the cell model, HepG2 cells were incubated with 10-7 mol/L insulin for 24 h to build insulin-resistant cell model.To evaluate the cells for insulin resistance, the cells were stimulated with fresh insulin for 24 h and the glucose uptake by these cells was carried out.The insulin-resistant cells were treated with different concentrations of FGF-21 for 24 h and insulin-treated cells were used as a control.The glucose uptake by the cells was detected by the method of glucose oxidizes/peroxides(GOD-POD);the synergy between insulin and FGF-21 was evaluated.The mRNA expression of GLUT1 in the insulin-resistant cells was detected by the real-time PCR.Glycogen synthesis of the cells was examined by the anthrone method.The results showed that HepG2 cells treated with 10-7 mol/L insulin for 24 h became resistant to insulin and the insulin resistance status was maintained for 48 h without change of cell morphology.FGF-21 could stimulate glucose consumption of the insulin-resistant model in a dose-dependent manner.The glucose consumption and glycogen synthesis of the insulin-resistant model were significantly improved by FGF-21 treatment.FGF-21 showed strong synergy with insulin in glucose uptake and glycogen synthesis of the model cells.While the cells became resistant to insulin, FGF-21 could increase the mRNA expression of GLUT1.Thus, It is concluded that FGF-21 stimulates glucose uptake in insulin resistant HepG2 cells through GLUT1 expression, stimulates glycogen synthesis and improves the glucose metabolism in the insulin resistant liver cell model.
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Objective In order to understand the differentially expressed genes and explore the effects on mechanism of gene expression induced by arsenic trioxide. Methods The mRNA was isolated from human HepG2 cells treated with arsenic trioxide( 5?mol/L ) and DMSO, respectively, then cDNA was synthesized. After restriction enzyme Rsa Ⅰ digestion, small sizes cDNA were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice, the DNA fragment was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after colony PCR. Results The forward subtracted cDNA library from HepG2 cell line induced by arsenic trioxide was successfully constructed. The sequencing analysis showed that there were eight clones contained ferritin H(L) chain in the library. Conclusion Arsenic trioxide can induce the up expression of ferritin H(L) chain protein in HepG2 cells, indicated that the ferritin H(L) chain may play certain role in the mechanism of anti-arsenical cytotoxicity in liver.
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Aim To establish insulin resistant HepG2 cell model induced by high concentration insulin in vitro culture and evaluate the effects of pioglitazone on insulin sensitivity and glucose metabolism in the cell model.Methods HepG2 cells were incubated with 5?10~(-7)mol?L~(-1) insulin for 16 h and insulin-stimulated()~3H-D glucose incorporation was determined.Then,insulin-stimulated glucose incorporation rate which was used to estimate insulin sensitivity of HepG2 cell,and glucose consumption,the amountsof glucose disappeared from the culture medium of HepG2 cells within 24 hours was determined.The insulin resistance cells were incubated with pioglitazone 1?10~(-5) mol?L~(-1).Results It was demonstrated that the incorporation rate of()~3H-D-glucose in insulin-resistant HepG2 cells was decreased significantly than that in the control cells.Insulin-resistant HepG2 cells were free from stimulation for 48 h by insulin,but the incorporation rate of()~3H-D-glucose was lower than that in control cells.Incubation of insulin resistance cells with pioglitazone significantly increased glucose uptake and glucose consumption by the cells compared with that of the control cells(P