【作者】 康顺爱；

【导师】 龚守良； 李校堃；

【作者基本信息】 吉林大学 ， 放射医学， 2009， 博士

【摘要】 紫外线(ultraviolet, UV)主要作用于皮肤,其中中波紫外线(ultraviolet B, UVB)主要通过生成活性氧(reactive oxygen species, ROS)自由基损伤表皮的角质形成细胞,导致细胞氧化应激反应,诱导细胞凋亡是UVB辐射氧化损伤细胞的最终结局。天然植物姜黄素(curcumin, Cur)的化学结构是由两个邻甲基化的酚和1个β-二酮组成,是多羟基酚类化合物,能够增加机体的抗氧化酶含量,如超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)等,这些抗氧化酶可通过增强机体的抗氧化能力清除ROS自由基,从而降低紫外线辐射引起的氧化损伤作用;Cur还具有防止膜脂质过氧化作用,维护线粒体膜电位(mitochondrial membrane potential,Δψ)的稳定性,阻止其释放细胞色素c(cytochrome c, Cyt c),通过调节凋亡相关蛋白如Bcl-2、Bax和caspase-3的表达,进而达到对机体的保护作用。本研究通过UVB辐照诱导人永生化角质形成细胞(HaCaT细胞)氧化损伤模型的建立,探讨Cur抗紫外线辐射损伤保护作用及其机制。实验结果表明,UVB辐照损伤HaCaT细胞的存活具有时间和剂量依赖性,即随着辐照剂量的加大和培养时间的延长,HaCaT细胞的存活率显著降低;细胞的凋亡率随着辐照剂量的加大而显著提高;UVB辐照后可显著增加细胞中的ROS、一氧化氮(nitric oxide, NO)和一氧化氮合酶(nitric oxide synthase, NOS)及脂质过氧化产物丙二醛(MDA)的含量,提高乳酸脱氢酶(LDH)的漏出量和胞浆游离钙离子浓度(concentration of free intracecellular calcium, [Ca2+]i),同时降低SOD和GSH-Px含量和线粒体膜电位(mitochondrial membrane potential,Δψ)。揭示了UVB辐照通过提高细胞氧化应激反应和脂质过氧化水平而破坏线粒体膜结构,从而增加了线粒体膜的通透性。同时,UVB辐照增加了促凋亡蛋白Bax、Cyt c和caspase-3的表达量,降低了抑凋亡蛋白Bcl-2的表达量,揭示了线粒体内凋亡相关蛋白的表达及其mRNA水平变化与凋亡发生之间的规律,通过启动线粒体凋亡途径和caspase-3凋亡途径,使细胞凋亡增加。UVB辐照HaCaT细胞后立即加入Cur,细胞的ROS、NO、NOS及MDA含量和LDH漏出量显著降低,并可显著提高HaCaT细胞的存活率和SOD及GSH-Px含量,表明Cur可抑制ROS自由基的生成和提高抗氧化酶活性,从而减轻细胞的氧化损伤;Cur可通过消除细胞周期阻滞,保持细胞周期进程的正常进行,促进细胞的生长;Cur能够提高Δψ和降低[Ca2+]i,维持HaCaT细胞线粒体膜电位的稳定及通透性,减少Cyt c蛋白漏出,降低促凋亡蛋白Bax和caspase-3的表达,增强抗凋亡蛋白Bcl-2表达,从而降低HaCaT细胞凋亡百分率。因此,Cur通过以上各因素的相互调节,达到对HaCaT细胞的保护作用。本研究为Cur的基础研究和临床应用及药物的开发利用提供了理论和实验依据,同时也进一步为辐射损伤的防护提供了重要的实验依据。更多还原

【Abstract】 Protective effects of curcumin against ultraviolet irradiation and its mechanismsUltraviolet (UV) is a species of electromagnetic waves which wave length is 200– 400 nanometer emitted from sun. It is divided into three frequency ranges according to different biological effect and ultraviolet wavelength that is Long-wave ultraviolet (UVA) which can penetrate into the epidermis and Midrange ultraviolet (UVB) which can penetrate skin for slight millimeters and Short-wave ultraviolet (UVC) which resides in vacuum. Most of UVB can reach to the ground because less is absorbed by ozonosphere and affect mainly the cuticulum. UVB is the source which results in skin sunburn and its injury potency on skin .It is 1000 times as much as UVA.It can lead to different level cytotoxicity through increasing ROS of skin tissue to cause oxidative stress reaction. The cells oxidative stress reaction play the key role in the pathogenesis in HaCaT cells.The effective research in skin by ultraviolet radiation is the focal point and hot spot in the shin research field at present. We can study protective effects against ultraviolet radiation through ant oxidative damage pathway.Keratinocytes are mainly the cells in forming epidermis, covering over 90 percent skin, and first contact directly the position to exogenous sensibiligen. It participates in various kinds of cytobiology procedure. If the multiplication, differentiation and abnormality of keratinocytes occurs, all kinds of disease can happen. Keratinocytes are the primary line of defense to resist UVR and the main target site of UVB energy. Ultraviolet radiation (UVR) of 95 percent is absorbed by keratinocytes. HaCaT cells belong to the cloned line of human being immortalization keratinocyte through spontaneous transformation cells after primary culture for long term and reserve complete differentiated capability of epidemic cell. HaCaT cells are the one of the most commonly used cells for researching keratinocyte apoptosis in the international. Ultraviolet B as irradiation photo source and HaCaT cells as irradiation cells, we have carried out research on protective effects of curcumin on HaCaT cells damaged by UV irradiation and its mechanisms.Curcumine (Cur) is the active constituent extracted from curcuma belongs to the traditional Chinese medicine. It is a kind of pigment of phenol and has on toxicity in normal cells. It can play many species of pharmacy-activities in anti-tumor, ant oxidation and protecting liver and so on based on protecting normal cell damaged by various kinds’harmful factors. Chemical constitution chains of Cur are unsaturated lipid families and aromatic series group with two orth-methylated phenols and oneβ-dike tone. Hydrogen contained in three groups of–OH、–OCH3 and–CH2 in molecular formula of Cur is active hydrogen, and these groups are the bioactive groups of anti-oxidation and so on. Its ant oxidation correlates intimately with distinct chemical constitution. Its phenol hydroxide radical can capture or get rid of free radical . So Cur regarded as a kind of natural antioxidant.Immune and molecular biological techniques used in the present study to examine the protective effects of Cur on the oxidation injury in HaCaT cells irradiated by UVB and its relative mechanisms. The results showed that under this experiment condition ,Cur decreased significantly the contents of ROS and lipid per oxidation product MDA, NO and NOS in HaCaT cells, decrease LDH leakage amount and plays a role in the ant oxidative stress. It can increase the antioxidase activity to enhance anti- free radical ability of HaCaT cells. At the same time, Cur improved the disintegration of mitochondria membrane potential (Δψ), reduced the Ca2+ concentration within the cells ([Ca2+]i), and maintained the cell membrane permeability and function. Cur suppressor cell apoptosis through decreasing Cyt c release from mitochondria in order not to activation caspase-3 which protected from the damage in HaCaT cells irradiated by UVB. Cur can raise survival rate and promote multiplication and sustain normal growth cycle through eliminate cell cycle arrest. The results in the present study still showed that Cur can block the corresponding oxidative damaged cell signaling to prevent the HaCaT cells from apoptosis through increasing the expressions of Bcl-2 gene and protein and decreasing the expressions of Bax, Cyt c and caspase-3 genes and proteins in the mitochondria and caspase two pathways. Cur can have protective effects on HaCaT cells by UVB irradiation.To sum up, that is Cur exerted its protection effects on oxidative injury in HaCaT cells apoptosis through the mutual roles of the above mentioned factors. The present study will offer the scientific theory and experimental bases for the protective effects of plants antioxidant on the damage irradiated by ultraviolet.1. Establishment of damage model on HaCaT cells irradiated by UVBCultured HaCaT cells were irradiated by UVB with the doses of 10, 20, 30, 40 and 50 mJ/cm2 and followed to culture for 6, 12, 18, 24, 48 and 72 h after irradiation in order to establish an injury model on HaCaT cells. The cellular viability and apoptosis detected by MTT. The results discovered that UVB irradiation inhibited the viability of HaCaT cells in a dose- and time-dependent pattern. As compared with that in the control group, the viability of HaCaT cells decreased significantly only in the 50 mJ/cm2 UVB group (P < 0.05) after cultured for 6 h; the viability of HaCaT cells decreased significantly only in the 40 and 50 mJ/cm2 UVB groups (P < 0.05, P < 0.01) after cultured for 12 h; the viability of HaCaT cells decreased significantly only in the 30 ~ 50 mJ/cm2 UVB groups (P < 0.01) after cultured for 18 h; the viability of HaCaT cells decreased significantly in all dose groups (P < 0.01) after cultured for 24– 72 h. It is hint that under this experiment condition, the UVB model were the 30 mJ/cm2 irradiation dose after cultured for 18 h..2. Effects of Cur on ROS content in HaCaT cells irradiated by UVBAs compared with that in the control group, ROS content in cultured HaCaT cells increased significantly after UVB irradiation with the doses of 30 mJ/cm2 (P < 0.01). HaCaT cells were treated with Cur of different doses immediately after irradiation by UVB and followed to culture for 18 h. As compared with that in the UVB model group, there was significant difference (P < 0.01) in ROS content in three Cur experiment groups with the pattern of Cur dose-effect, it means that ROS content decreased significantly with increasing concentration of Cur; but as compared with that in the control group, ROS content still increased significantly. It is hint that under this experiment condition, although Cur can play protective role in HaCaT cells irradiated by UVB through the decrease of ROS content, ROS content cannot come back to normal cellular level before irradiation.3. Effects of Cur on NO content and NOS activity in HaCaT cells irradiated by UVBAs compared with that in the control group, NO content and NOS activity in the cultured HaCaT cells increased significantly after UVB irradiation with the doses of 30 mJ/cm2 (P < 0.01). HaCaT cells were treated with Cur of different doses immediately after irradiation by UVB and followed to culture for 18 h. As compared with those in the UVB model group, there were significant difference in NO content and NOS activity (P < 0.01) in three Cur experiment groups with the pattern of Cur dose-effect. It means that NO content and NOS activity decreased significantly with increasing concentration of Cur. But as compared with those in the control group, there was not significant difference in NO conten only in the UVB + 5.0 mg/L Cur group,and there were significant difference in NOS activity in three Cur experiment groups(P < 0.01).It is hint that Cur can come back to normal cellular level before irradiation only in NO content in the 5.0 mg/L Cur concentration.NOS activity can not come back to normal cellular level before irradiation in all UVB + Cur groups.4. Effects of Cur on MDA and LDH contents in HaCaT cells irradiated by UVBAs compared with those in the control group, MDA and LDH contents in the cultured HaCaT cells increased significantly after irradiation by UVB with the doses of 30 mJ/cm2 (P < 0.01). HaCaT cells treated with Cur of different doses immediately after irradiation by UVB and followed to cultivat for 18 h. As compared with those in the UVB model group, there was significant difference in MDA contentin in three Cur experiment groups (P < 0.01) with the pattern of Cur dose-effect. It means that MDA content decreased significantly with increasing concentration of Cur. There was not significant difference in LDH content in UVB + 1.25 mg /L Cur group (P > 0.01) and there were significant decrease in LDH content in UVB + 2. 5 mg /L Cur group and UVB + 5.0 mg /L Cur group (P > 0.01). It is hint that under this experiment condition, Cur can suppressor cell membrane lipid per oxidation to protect cell membrane stability through cutting down MDA and LDH content. Cur can protect cell oxidative damage by UVB irradiation.5. Effects of Cur on antioxidative enzyme activities in HaCaT cells irradiated by UVBCultured HaCaT cells were irradiated by UVB with the doses of 30 mJ/cm2 and followed to culture for 18 h after irradiation. As compared with those in the control group, SOD and GSH-PX activities decreased significantly (P < 0.01). When HaCaT cells were treated with Cur of different doses immediately after irradiation by UVB and followed to culture for 18 h, as compared with those in the UVB model group, there was not significant difference in SOD and GSH-PX activities in the UVB + 1.25 mg/L Cur group (P > 0.01) and there were increase signification in the other groups. It show that under this experiment condition, Cur can play a protective role in HaCaT cells irradiated by UVB through enhance antioxidase activity.6. Effects of Cur on cell cycle progression and growth in HaCaT cells irradiated by UVBAs compared with that in the control group, survival rate of HaCaT cells irradiated by UVB irradiation decrease significantly. HaCaT cells were treated with Cur of different doses immediately after the doses of 30 mJ/cm2 UVB irradiation and followed to culture for 18h,as compared with that in UVB model group, survival rate increase all. Among the total groups, survival rate increased significantly only in UVB + 2.5 mg/L Cur groups and UVB + 5.0 mg /L Cur groups (P < 0.01). It is hint that under this experiment condition, Cur can promote growth effects after UVB irradiation, but it can not recover growth level pre-irradiation. For cell cycle, normal cell main distribute in G0/G1 and S stages. Cultured HaCaT cells were irradiated by UVB with the doses of 30 mJ/cm2 and followed to culture for 18 h after irradiation. As compared with those in the control group, there were not significant difference in the cell accounts of G0/G1 stage, there were decrease significant in the cell accounts of S stage and there were increase significant in the cell accounts of G2/M stage. HaCaT cells were treated with different Cur doses immediately after the doses of 30 mJ/cm2 UVB irradiation and followed to culture for 18h,as compared with that in UVB model group, there were not significant difference in the cell accounts of G0/G1 stage in all Cur concentration groups, and increase significant in that of S stage and decrease significant in that of G2/M stage(P < 0.01). It is hint that under this experiment condition,UVB irradiation can lead to cell cycle arrest through decrease the cell accounts of S stage and increase the cell accounts of G2/M stage. Cur can eliminate the cell cycle arrest to ensure normal cell cycle proceeding and maintain cell survival rate and promote cell multiplication. It is hints that under this experiment condition, Cur can not come back to normal cellular level of the cell accounts of G0/G1 and S stage pre-irradiation.7. Effects of Cur on apoptosis and necrosis in HaCaT cells irradiated by UVB The apoptosis and necrosis in HaCaT cells in this experiment detected with Annexin V-PI double dyes. The cultured HaCaT cells were irradiated by UVB with the doses of 30 mJ/cm2 and were added different Cur immediately separately and followed to culture for 18 h after irradiation. As compared with that in the UVB model group, the cell percentage of apoptosis and necrosis decreased significantly in three UVB + Cur groups (P < 0.01).Among the total, descended extent of apoptosis was more than that necrosis. As compared with that in the control group, that of apoptosis and necrosis increased significantly (P < 0.01). It is hint that under this experiment condition, Cur can protect effects through suppress cell apoptosis rate, but it can not come back to normal cellular level before irradiation.8. Effects of Cur onΔψand [Ca2+]i in HaCaT cells irradiated by UVBHaCaT cells were irradiated by UVB with the doses of 30 mJ/cm2 and followed to culture for 18 h after irradiation. Fluorescence Intensity ofΔψand [Ca2+]i increase significantly all(P < 0.01).It shows thatΔψdecrease significantly and [Ca2+]i increase significantly. When HaCaT cells were treated with different Cur doses immediately after irradiation by UVB and followed to culture for 18 h, as compared with those in the UVB model group,Δψdifference significantly (P < 0.01) with the pattern of Cur dose-effect, that isΔψincreased significantly with increasing concentration of Cur. Otherwise, [Ca2+]i decreased significantly only in the UVB + 2.5 mg /L Cur and the UVB + 5.0 mg /L Cur groups (P < 0.01),it was not difference significantly in the UVB + 1.25 mg /L Cur(P > 0.01). As compared with those in the control group, there were significant difference inΔψand [Ca2+]i in three UVB + Cur groups (P < 0.01).It is hint that under this experiment condition, Cur can protects the cell and stabilizes mitochondrial membrane through increasingΔψand decreasing [Ca2+]i,but it can not come back to normal cell level pre-irradiation.9. Effects of Cur on apoptosis relative protein expressions in HaCaT cells irradiated by UVBThe changes of caspase-3 depending on some genes and mitochondrial way in the apoptosis regulation were explored in the present experiment. Protein expressions were examined with flow cytometer and immunocytochemical method, mRNA levels was examined with RT-PCR methods. Cyt c, Bax and caspase-3 protein and their mRNA expressions in HaCaT cells were significantly higher and those of Bcl-2 gene expression was obviously lower in HaCaT cells irradiated by UVB (P < 0.01). When HaCaT cells were treated with different Cur doses immediately after irradiation by UVB and followed to culture for 18 h, as compared with those in the UVB model group,the proteins and their mRNAs level expressions of Cyt c, Bax and caspase-3 decrease significantly and Bcl-2 gene expression increase significantly (P < 0.01),Otherwise ratio of Bcl-2/Bax raise. It is thus clear that Cur can increase anti-apoptosis protein expression and decrease the promoting apoptosis protein expression to decrease apoptosis rate through regulating the two genes in apoptosis regulation pathways to block up corresponding signal conduction of oxidative damage for protecting effects in HaCaT by UVB irradiation.更多还原