ObjectiveTo investigate the effect of emodin on the expression of hypoxia inducible factor (HIF)-1α protein in rats with severe acute pancreatitis-associated renal injury and explore the possible mechanisms. MethodsA total of 72 rats were randomly divided into sham-operated group (n=24), severe acute pancreatitis with renal injury group (injury group, n=24), and treatment group (n=24). The sham-operated and injury groups were given 1.5 mL saline through intragastric administration before operation while the treatment group was fed with the same amount of 50 mg/kg emodin diluent. The pancreas and pancreatic tail-segment was dissociated and the head of pancreas was occluded in rats to form the model, and blood vessel forceps were loosed after three hours. All the rats were sacrificed 12, 24 and 36 hours after modeling. The level of ascites, serum amylase, creatinine, blood urea nitrogen were detected. Hematoxylin-eosin staining was used to observe the pancreatic and renal pathological changes, and immunohistochemical method was used to detect the expression of HIF-1α protein level in the kidney. ResultsCompared with the sham-operated group, the level of ascites, serum amylase, creatinine, blood urea nitrogen and the expression of HIF-1α protein level increased significantly. The tissue damage of pancreas and the kidney became more serious. Compared with the injury group, the kidney and pancreas function of the treatment group had a better performance. HIF-1α protein level significantly increased in the treatment group, and the difference had a statistical significance (P<0.05). ConclusionEmodin has a good protective effect on severe acute pancreatitis-associated renal injury. It may function through up-regulation expression of HIF-1α protein level to improve the ability of the kidney to tolerate hypoxia, and then reduce the cell apoptosis and necrosis of the kidney.
Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
ObjectiveTo explore the role and significance of hypoxia inducible factor lα (HIF-lα) and hypoxia microenvironment in the pathogenesis of post-traumatic heterotopic ossification by detecting the expression of HIF-lα in rat model of heterotopic ossification after Achilles tenotomy. MethodsA total of 140 male Sprague Dawley rats, aged 8-10 weeks, and weighing (210.1±10.6) g, were randomly divided into experimental group (n=70) and control group (n=70). In experimental group, the Achilles tendon was cut off and clamped to prepare post-traumatic heterotopic ossification model; in control group, only Achilles tendon was exposed. The general condition of rats was observed after operation, and at 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 days after operation, the Achilles tendon tissue was harvested from 6 rats for gross observation, histological observation, and immunohistochemical staining observation, and real-time fluorescence quantitative PCR and Western blot were used to detect the expressions of HIF-lα gene and protein at different time points in 2 groups. The X-ray films were taken and histological examination was done at 10 weeks after operation to evaluate the formation of heterotopic ossification. ResultsDuring the experiment, 1 rat died in experimental group at 3 days after operation, and the other rats survived to the end of the experiment. Gross and histological staining showed that the Achilles tendon had no obvious change, with normal tendon structure in control group at each time point. In experimental group, atrophy and necrosis of Achilles tendon stump were observed, with infiltration of inflammatory cells; and the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells. When compared with control group, the HIF-lα mRNA and protein expressions were significantly increased in experimental group at each time point (P < 0.05). Immunohistochemical staining showed that HIF-lα was positive in experimental group. According to the results of X-ray films and histological examination at 10 weeks after operation, heterotopic ossification was found in experimental group, but no heterotopic ossification in control group. ConclusionThe expression of HIF-lα significantly increases at early stage of post-traumatic heterotopic ossification after Achilles tenotomy, suggesting that the local hypoxia microenvironment plays an important role in the pathogenesis of heterotopic ossification.
Objective To investigate the effects of matrine on cell proliferation and expression of connective tissue growth factor( CTGF) and hypoxia inducible factor-1α( HIF-1α) of human lung fibroblast ( WRC-5) in normoxia ( 21% O2, 74% N2 , 5% CO2 ) and hypoxia ( 1% O2, 94% N2 , 5% CO2 )conditions. Methods MRC-5 cells were cultured and divided into differrent groups interfered with different dose of Matrine ( final concentration of 0 ～3. 2 mmol / L) in normoxia or hypoxia for 24 h. Cells were dividedinto 8 groups according to culture conditions, ie. normoxiagroup( N0 group) , normoxia + matrine 0. 2 mmol / L group( N0. 2 group) , normoxia + matrine 0. 4 mmol / L group( N0. 4 group) , normoxia + matrine 0. 8 mmol / L group( N0. 8 group) , hypoxia group( H0 group) , hypoxia + matrine 0. 2 mmol /L group( H0. 2 group) , hypoxia +matrine 0. 4 mmol /L group( H0. 4 group) , and hypoxia + matrine 0. 8 mmol / L group( H0. 8 group) . The MTT assay was used to measure the cell proliferation activity. Western-blot assay was used to examine the expression of CTGF and HIF-1α. Results Hypoxia promoted the cell proliferation in all groups( P lt;0. 05) .Matrine inhibited the proliferation of WRC-5 cells in a concentration-dependent manner in hypoxia or normoxia conditions( P lt;0. 05) . The expression of CTGF andHIF-1αwas lower in normoxia and higher in hypoxia( P lt;0. 01) . Matrine inhibited the expression of CTGF and HIF-1αin a concentration-dependent manner in hypoxiaand normoxia( P lt;0. 05) . Conclusion Matrine can inhibit the cell proliferation and the expression of CTGF and HIF-1αof WRC-5 cells in normoxia and hypoxia in a concentration-dependent manner.
ObjectiveTo observe the genes expression of hypoxia inducible factor 1α (HIF-1α) and HIF-2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. MethodsHigh density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2%O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type Ⅱ at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type Ⅱ, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. ResultsToluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group;extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type Ⅱ was positive in cytoplasm;when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t=8.345, P=0.001;t=7.683, P=0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P<0.05), but no significant difference was found at 3 weeks between the 2 groups (P>0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P<0.01). The mRNA expressions of collagen type Ⅱ and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P<0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P<0.05). ConclusionHIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.
Objective To investigate the influence of hypoxic preconditioning on pulmonary structure of rats exposed to simulated high altitude hypoxia and to explore the role of hypoxia inducible factor-1α（HIF-1α）.Methods Fifty-six Wistar rats were randomly divided into 7 groups(n=8 in each group),ie,a normal control group(N group)，an acute hypoxic control group(H0 group)，an acute hypoxic group(H1 group)，a 3 000 m hypoxic preconditioning group(C3.0 group)，a 3 000 m hypoxic preconditioning + acute hypoxic group (C3.1 group)，a 5 000 m hypoxic preconditioning group(C5.0 group)，and a 5 000 m hypoxic preconditioning + acute hypoxic group(C5.1 group).After treated with hypoxic preconditioning，the animals were exposed to simulated altitude of 6 000 m for 24 hours.Then the protein and mRNA expression of HIF-1α in lung of N，H0，C3.0 and C5.0 groups were assessed by Western blot and RT-PCR，respectively.The lung structure in N，H1，C3.1 and C5.1 groups was observed by light microscope and electron microscope.Results Pulmonary interstitial edema was apparently observed in H1 group，while significantly relieved in two hypoxic preconditioning groups.HIF-1α protein was not detected in rat lungs by Western blot analysis.Compared to N group，the levels of HIF-1α mRNA significantly increased in C3.0 group and C5.0 group(both Plt;0.01).Conclusions Hypoxic preconditioning can relieve hypoxic pulmonary interstitial edema and increase HIF-1α mRNA expression in rat lungs.HIF-1 may be involved in the process of hypoxic preconditioning in rat lungs.
ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． ResultsCompared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (Pgt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．
Objective To investigate the expression of hypoxia inducible factor 1α (HIF-1α) protein and the activation of phosphoinositid 3-kinase/Akt (PI3K/Akt) signal ing pathway in neurons under hypoxia ischemia condition,and to elucidate the role of PI3K/Akt on HIF-1α regulation in the developing neurons after hypoxia ischemia brain damage(HIBD). Methods Fifty-six SD rats aged 10 days were randomly divided into normal control group (n=12), sham operationgroup (n=12), experimental group (n=24), wortmannin treated group (n=4) and DMSO/PBS treated group (n=4). In theexperimental group, the rats were anesthetized with ethylether. The right common carotid artery was exposed and l igated. Then, they were exposed to hypoxia in a normobaric chamber filled with 8% oxygen and 92% nitrogen for 2.5 hours. In the sham control group, the right common carotid artery was exposed but was not l igated or exposed hypoxia. In the normal control group, the rats recevied no further processing. For wortmannin treated group and DMSO/PBS treated group, the rats received intraventricular injection of wortmannin or DMSO/PBS 30 minutes before hypoxia ischemia. The brain tissues were harvested from the rats in the normal control, sham operation and experimental groups at 4, 8 and 24 hours after hypoxia ischemia, but in the wortmannin and DMSO/PBS treated groups only at 4 hours. The HIF-1α protein expression and Akt protein expression were detected with immunohistochemistry method. HIF-1α, Akt and p-Akt protein expression were measured by Western blot analysis. Results In the experimental group, the HIF-1α expression was significantly increased at 4 hours after operation, reached the peak level at 8 hours, and began to decrease at 24 hours. The p-Akt protein was significantly increased at 4 hours, and began to decrease at 8 hours. However, the expression levels of HIF-1α and p-Akt protein in the normal control group were extremely low at each time point. So, the expression levels of HIF-1α in the experimental group was significantly higher than that in the normal control groups (P lt; 0.01), the expression of p-Akt protein in the experimental group at 4 and 8 hours was significant higher than that in the normal control group (P lt; 0.05). The change of Akt protein in the experimental group was not time-dependent, and no significant difference was evident when compared with that of the normal control group (P gt; 0.05). Using wortmannin, the PI3K/Akt specific inhibitor, HIF-1α protein expression was significantly decreased when compared with the DMSO/PBS treated group and experimental group (P lt; 0.01). Conclusion These results suggested that the HIBD of neonatal rats may activate PI3K/Akt signal ing pathway and further induce the expression of HIF-1α, indicating PI3K/Akt signal ing pathway and HIF-1α could be a potential target for treatment of neonatal HIBD.
To review the role of hypoxia inducible factor 1α (HIF-1α) in hypoxic-ischemic injury and its repair, and to analyze the possible mechanisms. Methods Recent l iterature on HIF-1α and its role in hypoxic-ischemic injury was reviewed and analyzed. Results HIF-1α was involved in the hypoxic-ischemic injury of various organs or tissues and their repair processes. Conclusion HIF-1α has a potential to treat common cl inical hypoxic-ischemic injuries and has a promisingfuture for appl ication.