| Hypertension is an important reason of high mortality of cardiovascular diseases.It can lead to target organs injury through a variety of mechanisms, such as heart, brain, kidney, liver and blood vessels. The antihypertensive treatment has important significance to reduce the incidence of cardiovascular events. One of the common points of the pathological and physiological mechanism of hypertension is that the bioavailability of active oxygen is increased, which is called the oxidative stress. Reactive oxygen species in the cardiovascular system regulate vascular endothelial function, which is involved in vascular endothelial inflammation, proliferation, migration, apoptosis, angiogenesis and other processes. The enhancement of ROS production and decline in the function of antioxidant system may lead to vascular endothelial injury and dysfunction, which is an important factor leading to cardiovascular diseases such as atherosclerosis and hypertension. At present, it is suggested that the repair of endothelial cells injury is regarded as the end point and the risk degree of hypertension treatment. Therefore, it is of great significance to study the injury and protection of vascular endothelial cells.Researchers found that anti-hypertensive drugs can lead to gene expression changesin kidney, bladder, heart, brain and liver tissue, in addition to the previously identified cardiovascular disease related genes, also including 16 genes which were not found in cardiovascular disease before, including interleukin-24(IL-24) gene, cathepsin Z(Ctsz) gene, and so on. Differentially expressed genes may be potential biomarkers as well as new targets for prevention and treatment of cardiovascular disease, which may provide valuable information for the development of cardiovascular disease therapy. As a novel tumor suppressor gene, IL-24 gene has obvious inhibitory effect on multiple tumor growth and metastasis.A series of molecular pathogenesis are common in cancer and cardiovascular disease. Increased toxic substances metabolic caused by smoking and dietary fat intake can cause the common molecular mechanisms related to these two kinds of diseases: oxidative stress and cell damage. Atherosclerosis may begin from the injury or infection of a single cell, or the transformation of a single arterial smooth muscle cell progenitor proliferation cloning, which is similar to the most widely held carcinogenic theory. The regulatory pathway of cell proliferation is involved in the progression of atherosclerotic plaque, vascular stenosis, restenosis after angioplasty and the development of cancer. The change of intercellular adhesion molecule is closely related to the formation of plaque, thrombosis, and tumor invasion and metastasis. Altered expression of the protease combined with thrombolytic therapy is involved in the expansion and bleeding of atherosclerotic plaques as well as in the invasion and metastasis of malignant tumors.Consider to the common pathogenesis of these two diseases, some novel therapeutic strategy can against both cardiovascular disease and cancer. Treatment strategies include the following: reducing oxidative stress injury; reducing cellular signals of chemokine, cytokines and growth factors by use of anti-inflammatory agents; inhibition of cell proliferation; anti-angiogenic to retard atherosclerosis plaque expansion and cancer infiltration metastasis; radiotherapy.In view of the biological effects of IL-24 gene related to many aspects of novel therapeutic strategies of cardiovascular disease and cancer, we hypothesized that it also has potential therapeutic role for the molecular pathogenesis of hypertension, and has close relationship with hypertensive disease occurrence and development, as well as antihypertensive drugs therapy. In the part of cell experiment, we investigated whether IL-24 gene has a protective effect against the endothelial injury induced by oxidative stress and the mechanism. Human umbilical vein endothelial cells(HUVECs) were exposed to increasing concentrations of H2O2 in the presence or absence of IL-24, which was introduced via Lipofectamine® 2000-mediated transfection. The successful uptake of the IL-24 plasmid was confirmed by RT-PCR at 24 h post-transfection. The effects of H2O2 and IL-24 on the proliferation and migration of the HUVECs was determined using cell migration assays. Cell viability was determined using Cell Counting Kit-8(CCK-8). Apoptosis and the measurement of the intracellular reactive oxygen species(ROS) levels were determined by flow cytometry, and the levels of caspase-3, which is associated with apoptosis, were determined by western blot analysis. PI3K/PKB signaling pathway proteins expression was detected by Western-blot method, and the activation of the pathway has a significant antioxidant activity, and is involved in cell proliferation and apoptosis.Real-time PCR and western blot analysis were also used to measure the levels of multiple cardiovascular disease-associated factors. In vivo experiments were also performed using a rat model of hypertension which was constructed by angiotensin II infusion using an osmotic pump. The m RNA and protein levels of IL-24 were measured in both the control and hypertensive rats; the effects of treatment with enalapril and nifedipine on the IL-24 levels were also examined.Our results revealed that IL-24 protected against the H2O2-mediated abnormal increase in HUVEC proliferation. IL-24 also antagonized H2O2 by reducing the content of ROS in the cells, thus decreasing cellular oxidative damage, improving the cellular survival rate, reducing apoptosis, reducing the protein expression of cleaved caspase-3, and decreasing the expression of cardiovascular disease-related factors. IL-24 may activate the PI3K/PKB signaling pathway to exert antioxidant effect, and then affect its downstream signaling pathways such as NF-κB and AP-1 signaling to regulate the vascular endothelial function and cardiovascular disease complications. The results from our in vivo animal experiments revealed that IL-24 expression was lower in the hypertensive rats compared to the healthy controls. Additionally, the IL-24 levels increased following anti-hypertensive therapy. IL-24 is closely related to the occurrence and development of hypertension.In summary, IL-24 gene can inhibit the abnormal proliferation of vascular endothelial cells, reduce oxidative stress, play the role of anti-oxidative damage and anti-apoptosis, reduce the protein expression of cleaved caspase-3, improve cell survival rate, and activate the PI3K/PKB signaling pathway to exert antioxidant effect. At the same time, IL-24 can down regulate the expression of genes related to cardiovascular diseases, and reduce the incidence of cardiovascular diseases. IL-24 may be involved in the molecular mechanism of enalapril and nifedipine treatment, suggesting that IL-24 gene may be the new targets and the key gene of molecular pathways of antihypertensive therapy. However, the detailed mechanism of IL-24 involved in antihypertensive drug therapy still needs further study.Taking into account the common pathogenesis of cancer and cardiovascular disease and the inhibitory effect of IL-24 on the pathogenesis induced by ROS, IL-24 gene may provide basic treatment strategies for various cardiovascular diseases caused by ROS.Part 1 Effect of IL-24 gene against the oxidative damage in HUVECs caused by hydrogen peroxide Objective:To investigate the protective effect of IL-24 on H2O2 induced vascular endothelial injury and the possible mechanism, and the correlation between IL-24 and cardiovascular disease. Methods:HUVECs were grown in DMEM at 37℃ in a humidified atmosphere containing 5% CO2. The experimental operations were performed ingood condition of cell growth. Cell experiment groups: â… Control group Normal cell culture, containing 10% fetal bovine serum cultured in DMEM medium; â…¡H2O2 damage group Each culture hole was added with a final concentration of 0.10 and 0.30 mmol/L H2O2 respectively, as the lower and the higher concentration; â…¢ H2O2 damage+IL-24 protection group The operation of the cell transfection was carried out before the injury.The concentration of H2O2 same with group â…¡,the recombinant plasmid p DC316-h IL-24 mixed with liposome Lipofectamine 2000 10μl before transfection, the specific operation in accordance with the Lipofectamine 2000 specification, theratio of recombinant plasmid p DC316-h IL-24 and liposome(μg:μl=1:1); IV H2O2 damage+empty plasmid group The concentration of H2O2 same with groupâ…¡,the ratio ofempty plasmid p DC316 and liposome(μg:μl=1:1), transfection methods like before; V The ratio of recombinant plasmid p DC316-h IL-24 and liposome same with group â…¢; VI The ratio of empty plasmid p DC316 and liposome same with group IV. Cell density in group â…¢-VI reached about 40-50%, then IL-24 gene transfection, and after 24 hours for RT-PCR detection, confirmed the transfection efficiency, after 48 hours were added to the respective concentrations of hydrogen peroxide for injury. Cells were collected after the intervention. The inhibitory effect of IL-24 on abnormal proliferation and migration of cells was detected by migration test, the CCK-8 method of cell activity, cell apoptosis was detected by flow cytometry, and the levels of caspase-3, which is associated with apoptosis, were determined by western blot analysis. Intracellular ROS levels were determined by flow cytometry and fluorescence probe labeling, and PI3K/PKB signaling pathway protein expression was detected by Western-blot method. Real-time PCR and Western-blot method for cardiovascular disease associated factor RNA and protein expression detection. Results:IL-24 can obviously inhibit the abnormal cell proliferation induced by low concentration H2O2, compared with the H2O2 group and H2O2+IL-24 group, the difference between the two groups was statistically significant, P<0.05; IL-24 can reduce the content of ROS in cells.ROS content in H2O2 injury group was significantly higher than other groups, compared with the control group and IL-24 protection group, the differenceswere statistically significant, P<0.001; IL-24 protected cells against oxidative damage, and it can significantly reduce the cell apoptosis and the protein expression of cleaved caspase-3. H2O2 damage group and IL-24 protection group were with statistically significant differences, P<0.001; cell survival rate was the lowest in H2O2 group, with significant difference compared to the control group, P<0.001. IL-24 protection can significantly reduce cell injury and death induced by oxidative stress, and cell survival rate increased significantly. Compared with H2O2+IL-24 group, the H2O2 group had obvious statistical difference, P<0.05; Protein expression of PI3K/PKB(Akt) signaling pathway in H2O2 injury group were increased, compared with the control group, the difference was statistically significant, P<0.05. In the case of IL-24 protection, it can further increase the expression of signaling protein, with a significant difference, P<0.001, so as to further play the role of antioxidant; IL-24 can down-regulation the expression of cardiovascular disease related factors Angiotensinogen, endothelin-1, ATRAP, and PDGF stimulated by H2O2, P<0.001. Conclusion:As a new therapeutic target for cardiovascular disease, IL-24 can inhibit the abnormal proliferation and migration of vascular endothelial cells, improve the survival rate of cells, play the effectof anti oxidative stress and anti apoptosis, reduce the protein expression of cleaved caspase-3, and activate the PI3K/PKB signaling pathway to exert antioxidant effect. At the same time, IL-24 can downregulate the increased cardiovascular diseases relevant genes expression after vascular injury, reduce the occurrence of cardiovascular disease. Taking into account the commonality of cancer and cardiovascular disease, and IL-24 inhibition effect on variety of pathogenesis caused by ROS, IL-24 gene may provide a treatment strategy for vascular diseases. Part 2 Correlation between IL-24 gene and the development and treatment of hypertension Objective:To investigate the correlation between IL-24 gene and the development and antihypertensive medication of hypertension. Methods:Normal SD rats were divided into control group(n=12)andhypertensive model group(n=36). Establishment of hypertensive rat model by using angiotensin II micro osmotic pumps. Dosage calculation was according with the instructions of micro osmotic pump and the weight of rats(500 ng/(kg·min)).The medicated micro osmotic pump was placed underthe abdominal skin of rats through operation and with sterile suture. The survival state, mental condition and blood pressure of rats were observed daily. The control group(n=12) and some rats in hypertensive model group(n=12) were sacrificed 1 week after they experienced a spike in blood pressure. The heart and kidney tissue were harvested from these animals, and real-time PCR and western-blot were performed to detect the expression of IL-24. The remaining hypertensive rats were divided into two groups, treatment group(n=12) and untreated group(n=12).The rats in treatmen group were administered enalapril(35mg/Kg/d) and nifedipine(30mg/Kg/d) orally.The rats were killed 1 week after stable blood pressure decreased, with the same method of detection of IL-24 gene expression in RNA and protein level. Results:IL-24 gene RNA and protein levels in heart and kidney tissue were detected by Real-time PCR and western-blot in successful hypertensive rat model and after antihypertensive therapy. The results showed that after the successfulconstruction of hypertension modeling, IL-24 gene expression was significantly reduced, compared with the control group with significant difference. After antihypertensive therapy, the expression of IL-24 gene was significantly increased, compared with the untreated group had obvious difference, P <0.001. The western-blot results showed that the IL-24 protein expression of successful hypertension model rats decreased obviously, compared with the control group, compared with the β-actin, IL-24 protein expression levels were(0.95±0.041/1.87±0.062), P<0.001, with statistically significant difference between the control rats and hypertensive rats. After antihypertensive medication, IL-24 protein expression was up-regulated, compared with the untreated group, compared with the β-actin, IL-24 protein expression levels were(1.54±0.014/0.81±0.042), P<0.001. Compared with antihypertensive treatment group, untreatment group has statistics significance difference. Conclusion:By constructing the hypertension rat model, it has proved that IL-24 gene RNA and protein expression were closely associated with the occurrence and development of hypertension. Gene expression decreased after elevated blood pressure, while antihypertensive medication can lead to increased gene expression. All these results suggested that IL-24 may be involved in the molecular mechanism of enalapril and nifedipine treatment. However, the detailed mechanism of IL-24 involved in antihypertensive medication still needs further study. IL-24 gene may be the new targets and the key gene of molecular pathways in hypertension treatment. |
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