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 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.
Objective Ginsenoside Rg1 could increase the tolerance of neural hypoxia and ischemia under stress, and play an anti-apoptotic effect in hypoxia ischemia brain damage (HIBD). To investigate the effects of ginsenoside Rg1 on neural apoptosis and recovery of neurological function in neonatal rats with HIBD, and to explore the possible mechanism. Methods Fifty-four 10-day-old SD rats (weighing 16-22 g) were randomly allocated into sham-operation group (Sham group, n=6), HIBD model group (HIBD group, n=24), and ginsenoside Rg1 treatment group (Rg1 group, n=24). SDrats in HIBD group and Rg1 group were made the models of HIBD by l igation of the right common carotid artery (CCA) and subsequently hypoxic ventilation (8%O2 plus 92%N2) for 2.5 hours; and in Sham group, the right CCA was only exposed without l igation of CCA and hypoxic ventilation. Intraperitoneal injection of 0.1 mL normal sal ine (NS) containing 40 mg/kg Rg1 was given immediately after operation in Rg1 group, intraperitoneal injection of 0.1 mL pure NS was given in both HIBD group and Sham group and was repeated every 24 hours. The general state of SD rats was monitored after operation, and Longa scores were recorded to evaluate the neurological function at 4, 8, 24, and 72 hours after HIBD. Western blot and immunohistochemistry staining were used to detect protein expressions of both hypoxia inducible factor 1α (HIF-1α) and cleaved caspase 3 (CC3). TUNEL staining was used to evaluate neural apoptosis in situ. Results All rats survived to the end of the experiment. Neurological dysfunction was observed in both HIBD group and Rg1 group, showing significant difference in Longa score when compared with that in Sham group (P lt; 0.05). There was significant difference in Longa score between Rg1 group and HIBD group at 72 hours after HIBD (P lt; 0.05). Western blot showed that the protein expressions of both HIF-1α and CC3 were observed at every time point in every group. The expressions of HIF-1α protein in HIBD group and Rg1 group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P lt; 0.05), and the expressions in Rg1 group were significantly higher than those in HIBD group (P lt; 0.05). The expressions of CC3 protein in HIBD group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P lt; 0.05), and significant difference was found between Rg1 group and Sham group only at 4 hours (P lt; 0.05). Immunohistochemistry staining demonstrated that HIF-1α and CC3 protein mainly distributed in nucleusand cytoplasma, the results of HIF-1α and CC3 protein expression were similar to the results by Western blot. TUNEL staining showed that the positive cells were characterized by yellow or brown particle confined within nucleus. The number of apoptotic cells at every time point in HIBD group was significantly higher when compared with that in Sham group (P lt; 0.05), and the number of apoptotic cells in Rg1 group was significantly lower when compared with that in HIBD group at 8, 24, and 72 hours (P lt; 0.05). Conclusion Rg1 could inhibit Caspase 3 activation by strengthening and stabil izing HIF-1α signal pathway, and plays a role of anti-apoptosis in neonatal rats with HIBD.
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 relationship between the expression of hypoxia inducible factor 1α (HIF-1α) and the neuron apoptosis during a hypoxia ischemia brain damage and explore the role of HIF1α in regulating the neuron apoptosis and repairing the brain damaged by hypoxia and ischemia. Methods Forty SD rats aged 10 days were randomly divided into the experiment group and the control group, with 20 rats in each group. In the experimental group, the rats were anesthetized with ethylether. The right common carotid artery was exposed and ligated. Then, they were exposed to hypoxia ina normobaric chamber filled with 8% oxygen and 92% nitrogen for 2.5 hours. In the control group, the right common carotid artery was exposed but was not ligated or exposed to hypoxia. The brain tissues were harvested from the rats in the both groups at 4, 8, 24, 48 and 72 hours after the hypoxia and ischemia, and fromthe rats in the control group at the same time points. The HIF-1α protein expression and the cleaved caspase 3 (CC3) protein expression were detected with the immunohistochemistry method. The apoptosis cells were detected with the TUNEL staining method. Results In the experimental group, the HIF-1α expression was significantly increased at 4 hours after operation, at the peak level at 8 hours, and began to decrease at 24 hours. The CC3 protein was expressed at 4 hours after operation, and was slightly expressed at 8 hours, but was significantly increased at 24 hours; the higher levels were maintained at 48 and 72 hours. However, in the control group, both the expression levels of HIF-1α and the CC3 protein were extremely low. So, the expression levels of HIF-1α andthe CC3 protein were significantly higher in the experimental group than in the control group (P＜0.01). The TUNEL staining showed that in the experimentalgroup the positive cells were significantly increased after the hypoxia and ischemia, with a peak level at 72 hours after the hypoxia and ischemia; however, in the control group there were few positive cells.TUNEL positive cells in the experimental group were significantly more than that in the control group(P＜0.01).ConclusionThe expression tendency of HIF-1α is completely different from that of CC3.HIF-1α may have a protective role in regulating the neuron apoptosis in the neonatal hypoxia-ischemia brain damage and may promote the repairing and rebuilding process in the brain that was damaged by hypoxia and ischemia.
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).
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.
ObjectiveTo summarize the research progress of the regulation effect of hypoxia inducible factor (HIF) on intervertebral disc. MethodsThe domestic and foreign related literature about the regulation effect of HIF on intervertebral disc was reviewed, summarized, and analyzed. ResultsHIF is a key transcription factor that is in response to hypoxia by cells, which is widely distributed in tissues and organs, including intervertebral disc. Hypoxia inducible factor is expressed highest in the nucleus pulposus which has the lowest oxygen concentration in the intervertebral disc. The effects of HIF include the regulation of nucleus pulposus differentiation and development, maintenance of the survival, energy metabolism, and anabolism of nucleus pulposus cells, and maintenance of the stability of extracellular matrix. ConclusionHIF plays a vital role in the development and differentiation of intervertebral disc and maintenance of physiological function, which may become a target for the research of the mechanism and the treatment of intervertebral disc degeneration.
Objective Isoflurane has an acute preconditioning effectiveness against ischemia in kidney, but this beneficial effectiveness can only last for 2-3 hours. To investigate whether isoflurane produces delayed preconditioningagainst renal ischemia/reperfusion (I/R) injury, and whether this process is mediated by hypoxia inducible factor 1α(HIF- 1α). Methods A total of 52 male C57BL/6 mice were randomly assigned to 4 groups (n=13 in each group): the controlgroup (group A), PBS/isoflurane treated group (group B), scrambled small interference RNA (siRNA)/isoflurane treated group (group C), and HIF-1α siRNA/isoflurane treated group (group D). In groups C and D, 1 mL RNase-free PBS containing 50 μg scrambled siRNA or HIF-1α siRNA was administered via tail vein 24 hours before gas exposure, respectively. Equivalent RNasefree PBS was given in groups A and B. Then the mice in groups B, C, and D were exposed to 1.5% isoflurne and 25%O2 for 2 hours; while the mice in group A received 25%O2 for 2 hours. After 24 hours, 5 mice in each group were sacrificed to assesse the expressions of HIF-1α and erythropoietin (EPO) in renal cortex by Western blot. Renal I/R injury was induced with bilateral renal pedicle occlusion for 25 minutes followed by 24 hours reperfusion on the other 8 mice. At the end of reperfusion, the serum creatinine (SCr), the blood urea nitrogen (BUN), and the histological grading were measured. Results The expressions of HIF-1α and EPO in groups B and C were significantly higher than those in group A (P lt; 0.01). The concentrations of SCr and BUN in groups B and C were significantly lower than those in group A, as well as the scores of tubules (P lt; 0.01), and the injury of kidney was amel iorated noticeably in groups B and C. The expressions of HIF-1α and the concentrations of SCr and BUN in group D were significantly lower than those in group A (P lt; 0.01). Compared with groups B and C, the expression of HIF- 1α and EPO in group D decreased markedly (P lt; 0.01), the concentrations of SCr and BUN were increased obviously, as well asthe scores of tubules (P lt; 0.01), and the renal injury was aggratived significantly. Conclusion Isoflurane produces delayed preconditioning against renal I/R injury, and this beneficial effectiveness may be mediated by HIF-1α.
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.