自噬在维持急性冷暴露大鼠肝脏能量代谢中的作用

发布时间：2018-03-11 00:25

本文选题：冷暴露　切入点：肝组织　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：【背景】冷暴露是常见的特殊环境暴露因素。我们以往的研究发现，-15℃急性冷暴露0.5h可以引起大鼠肝脏能量水平的改变及相关细胞通路活性的变化，复方制剂可以显著缓解急性冷暴露大鼠体温下降，但其机制仍不清楚。自噬是细胞正常生理过程，可以清除损伤细胞器，提供能量代谢底物并抑制凋亡发生；能量应激情况下，自噬可以维持细胞ATP水平。我们设想急性冷暴露能否引起肝脏细胞自噬水平改变及其产生的影响，复方制剂能否影响急性冷暴露大鼠肝脏能量水平及自噬是否参与复方制剂提高大鼠耐寒能力的过程值得探讨。【目的】通过建立大鼠急性冷暴露模型，观察急性冷暴露对大鼠肝细胞能量水平及细胞凋亡的影响，探讨自噬水平变化产生的影响及调控机制，并进一步探讨自噬在复方制剂调控急性冷暴露条件下大鼠肝细胞能量水平及细胞凋亡中的作用及机制。为阐明急性冷暴露对机体造成的损伤、急性冷暴露过程中机体能量的调控及复方制剂提高耐寒能力的作用机制提供理论依据，为冷暴露条件下的机体防护措施提供理论基础。【方法】 1.急性冷暴露模型的建立：雄性SD大鼠230g±10g体重，独立暴露于-15℃低温试验舱，暴露时间为4h。 2.肝脏糖原含量测定：用肝糖原检测试剂盒测定大鼠肝组织肝糖原水平。 3.肝脏细胞能量水平测定：用ATP检测试剂盒，通过多功能酶标仪进行发光度测定并得到大鼠肝细胞ATP含量值。 4.凋亡水平检测：用western blot方法测定肝细胞凋亡相关蛋白caspase-3活化水平及TUNEL染色法观察TUNEL阳性细胞。 5.自噬水平检测：用western blot方法测定肝细胞自噬标志物LC3-I和LC3-II的表达变化；用免疫组化方法，通过MDC染色方法进行肝细胞自噬水平的检测。 6.自噬抑制剂对机体的影响：采用腹腔注射的方法，降低自噬水平，同时观察其对肝组织冷暴露应激状态下能量代谢可能产生的影响。 7.复方制剂对机体的影响：采用连续灌胃方法，持续灌胃3天。观察能量及自噬水平的变化。【结果】 1.急性冷暴露对大鼠肝细胞凋亡水平的影响 -15℃急性冷暴露4h引起大鼠肝细胞凋亡增加。急性冷暴露组大鼠肝细胞caspase-3剪切水平显著增加，TUNEL阳性细胞数显著增加（p＜0.05）。 2.急性冷暴露对大鼠肝脏ATP及肝糖原水平的影响 -15℃急性冷暴露4h引起大鼠肝脏细胞ATP、肝糖原水平显著下降。室温对照组大鼠肝糖原5.03±0.86mg/g，而冷暴露对照组大鼠肝糖原降至2.24±1.03mg/g；对照组大鼠ATP水平为0.293±0.018μmol/mg，冷暴露4h组大鼠ATP水平降低至0.195±0.044μmol/mg。两者均具有统计学意义（p＜0.05）。 3.急性冷暴露对肝脏自噬水平的影响 MDC染色检测发现，-15℃急性冷暴露4小时引起肝细胞自噬泡数量增加，与对照组相比具有统计学意义（p＜0.05）；WB检测发现LC3-II灰度水平在急性冷暴露后显著升高（p＜0.05）。提示急性冷暴露引起大鼠肝脏自噬水平显著增加。 4.自噬抑制剂对急性冷暴露大鼠肝脏细胞凋亡水平、能量水平及自噬水平的影响腹腔注射自噬抑制剂渥曼霉素可以显著降低肝细胞自噬水平（p＜0.05），渥曼霉素引起急性冷暴露大鼠肝细胞凋亡水平显著升高，进一步增加caspase-3蛋白的剪切及TUNEL染色阳性细胞数量（p＜0.05），并引起肝细胞ATP含量显著降低，冷暴露抑制剂组大鼠ATP水平为0.146±0.018μmol/mg与冷暴露对照组大鼠ATP水平0.193±0.047μmol/mg相比具有统计学意义（p＜0.05）。而冷暴露抑制剂组大鼠肝糖原水平与冷暴露对照组大鼠相比无明显差异。 5.复方制剂对急性冷暴露大鼠肝细胞凋亡水平、肝脏能量水平及肝细胞自噬水平的影响复方制剂灌胃3天可以显著降低急性冷暴露大鼠肝细胞凋亡水平（p＜0.05），并提高急性冷暴露大鼠的肝脏细胞ATP及肝糖原水平，并同时降低肝组织的自噬水平。冷暴露复方制剂组的大鼠ATP水平为0.236±0.018μmol/mg，与冷暴露对照组大鼠ATP水平0.187±0.030μmol/mg相比具有统计学意义（p＜0.05），冷暴露复方制剂组大鼠肝糖原2.20±1.24mg/g，而冷暴露对照组大鼠肝糖原降至1.38±0.97mg/g，相比具有统计学意义（p＜0.05）。【结论】 1.急性冷暴露可以引起大鼠肝细胞凋亡，，肝细胞能量水平降低，并引起细胞自噬水平增加。 2.寒冷诱导的自噬有助于维持一定的ATP和代谢水平并抑制凋亡的发生。 3.复方制剂可以部分逆转急性冷暴露诱导增加的自噬水平，同时减少细胞凋亡水平并提高急性冷暴露大鼠肝细胞ATP水平以及肝糖原水平。提示复方制剂能够在一定程度上减轻急性冷暴露对肝脏造成的损伤。
[Abstract]:[background]
Cold exposure is a common specific environmental exposure factors. Our previous study found that -15 C acute cold exposure 0.5h can change caused by the change of rat liver energy level and related cell pathway activity, the compound preparation can significantly relieve the acute cold exposure of rats decreased body temperature, but the mechanism is still unclear. Autophagy is normal process, can the clearance of damaged organelles, energy metabolism substrate and inhibiting apoptosis; energy stress, autophagy can maintain cellular ATP level. We assume that acute cold exposure can cause liver cell autophagy level change and influence of compound preparation can influence the acute cold exposure of rat liver energy level and autophagy is involved in the process improve the cold resistance ability of rat compound preparation is worth exploring.
[Objective]
Through the establishment of acute cold exposure model rats, observe the effect of acute cold exposure on the energy level of liver cells and cell apoptosis in rats, to explore the effect of autophagy level changes and regulation mechanism, and to further explore the regulation of autophagy in compound acute cold exposure effect and mechanism of apoptosis of liver cells and the level of energy under the condition of the cells of rats on the body caused by injury. In order to elucidate the acute cold exposure, acute cold exposure mechanism to improve cold tolerance control and compound body energy in the process to provide a theoretical basis for cold exposure under the condition of body anti theoretical foundation support measures.
[method]
1. the establishment of acute cold exposure model: the male SD rats were 230g + 10g body weight and were exposed to -15 centigrade cryogenic test compartment independently, the exposure time was 4h.
2. determination of liver glycogen content: Determination of liver glycogen level in rat liver tissue with liver glycogen detection kit.
3. determination of liver cell energy level: detection kit by ATP, measurement of luminosity by multi-function enzyme labeling and ATP content of rat liver cells.
4. detection of apoptosis level: the activation level of hepatocyte apoptosis related protein caspase-3 was measured by Western blot method and TUNEL positive cells were observed by TUNEL staining.
5. detection of autophagy level: the expression of LC3-I and LC3-II of hepatocyte autophagy markers were detected by Western blot method. The autophagy level of hepatocytes was detected by immunohistochemical staining and MDC staining.
6., the effect of autophagy inhibitor on the body: the method of intraperitoneal injection can reduce the level of autophagy, and observe its effect on the energy metabolism of liver tissue under cold exposure stress.
7. the effect of compound preparation on the body: continuous gavage method and continuous gavage for 3 days. The changes of energy and autophagy were observed.
[results]
The effect of 1. acute cold exposure on the level of apoptosis in rat liver cells
Acute cold exposure at -15 C increased 4H hepatocyte apoptosis in rats. In acute cold exposure group, the caspase-3 level of liver cells increased significantly, and the number of TUNEL positive cells increased significantly (P < 0.05).
The effect of 2. acute cold exposure on the level of liver ATP and liver glycogen in rats
-15 C acute cold exposure induced by 4H in liver cells of ATP rats, liver glycogen level was significantly decreased at room temperature. The control group of rat liver glycogen was 5.03 + 0.86mg/g, while the control group cold exposure of rat liver glycogen decreased to 2.24 + 1.03mg/g; ATP level of the control group rats was 0.293 + 0.018 mol/mg, ATP level of 4H group exposed to cold rats decreased to 0.195 + 0.044 u mol/mg. both were statistically significant (P < 0.05).
3. effect of acute cold exposure on the level of autophagy in the liver
MDC staining, -15 C acute cold exposure 4 hours caused by global increase in the number of liver cell autophagy, compared with the control group with statistical significance (P < 0.05); WB detected LC3-II gray level significantly increased in acute cold exposure (P < 0.05). Acute cold exposure induced autophagy inrat liver increased significantly.
The effect of 4. autophagy inhibitors on the level of apoptosis, energy level and autophagy in acute cold exposed rats
Intraperitoneal injection of kanamycin fermentation Manchester autophagy inhibitors can significantly reduce the hepatic autophagy level (P < 0.05), Ottawa Manchester kanamycin induced acute cold exposure level of apoptosis of rat liver cells increased significantly, further increase the shear and TUNEL staining of caspase-3 protein positive cells (P < 0.05), and liver cells caused by ATP was significantly decreased that cold exposure level of ATP inhibitor group rats was 0.146 + 0.018 mol/mg and cold exposure control ATP group rats compared to 0.193 + 0.047 mol/mg was statistically significant (P < 0.05). But the cold exposure level of glycogen and cold rats exposed to inhibitors of the rats in the control group had no significant difference.
Effect of 5. compound preparation on the level of hepatocyte apoptosis, liver energy level and the level of autophagy of liver cells in rats with acute cold exposure
The intragastric administration of compound preparation for 3 days can significantly reduce the level of apoptosis of liver cells in rats of acute cold exposure (P < 0.05), and improve the rat liver ATP cells and liver glycogen levels of acute cold exposure, and at the same time reduce the level of autophagy in liver tissue. Cold exposure compound group level of ATP rats was 0.236 + 0.018. Mol/mg, and cold exposure compared to 0.187 + 0.030 mol/mg was statistically significant ATP level of the control group of rats (P < 0.05), cold exposed rats liver glycogen preparation of compound 2.20 + 1.24mg/g, while the control group cold exposure of rat liver glycogen decreased to 1.38 + 0.97mg/g, compared with statistical significance (P < 0.05).
[Conclusion]
1. acute cold exposure can induce apoptosis of hepatocytes in rats, decrease the level of hepatocyte energy, and increase the level of autophagy.
2. cold induced autophagy helps to maintain a certain level of ATP and metabolism and inhibit the occurrence of apoptosis.
3. compound autophagy induced the increase of exposure can partially reverse the acute cold, while reducing the level of apoptosis and improve acute cold exposure level of ATP rats liver cells and liver glycogen level. Suggesting that compound preparation could reduce the liver damage caused by acute cold exposure in a certain extent.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】