- 學位論文
香椿葉萃取物對於盲腸結紮穿刺誘發C57BL/6小鼠敗血症之氧化性傷害的影響評估
Effects of Toona sinensis leaf extracts on the oxidative stress in the C57BL/6 mice with cecal ligation and puncture-induced sepsis
摘要
研究背景與目的：敗血症為患者受到感染造成發炎反應並導致組織損傷之疾病，也是住院病患常見的致死病因，先前的研究發現，敗血症的病程發展與氧化壓力增加有關。香椿為一種可以當中藥材或是食材服用的植物，有研究指出，香椿具有抗氧化的特性，因此本篇研究擬探討香椿葉萃取物對於敗血症小鼠血液及肝組織在氧化傷害方面的影響。 實驗方法：本篇研究以C57BL/6小鼠進行盲腸結紮穿刺手術 (cecal ligation and puncture, CLP)來誘發敗血症，手術前先給予小鼠香椿葉萃取物 (Toona sinensis leaf extracts, TS, 100mg/kg/day) 4週後再進行CLP。本實驗將小鼠分成五組：對照組 (Control)、香椿葉萃取物組 (TS)、假手術組 (Sham)、盲腸結紮穿刺組 (CLP)、盲腸結紮穿刺補充香椿葉萃取物組 (CLP + TS)。於手術後各0、3、6、9、12、15小時犧牲小鼠，取其血液和肝組織進行本實驗，血液實驗項目包含了：血液常規檢查 (CBC)、肝功能指標 (AST、ALT、ALP)、抗氧化指標(GSH、SOD)。肝組織實驗項目包括：抗氧化指標 (GSH)、氧化傷害指標 (NO)以及與氧化傷害有相關的蛋白質表現 (iNOS、HMGB1)。 實驗結果：與Control組比較，TS組之血液白血球數量及肝組織HMGB1表現明顯減少，肝功能、血液及肝組織抗氧化物以及氧化傷害等皆沒有顯著差異。Sham組和CLP組兩者於手術後，相同時間點的比較：於術後0小時，CLP組的血液及肝組織GSSG濃度顯著大於Sham組，而血液GSH濃度、GSH/GSSG比例則顯著小於Sham組；於術後3小時，CLP組的RBC數量、HGB濃度、淋巴球比例、血液total GSH濃度、iNOS及HMGB1表現量顯著大於Sham組，而嗜中性球比例、肝組織GSH及total GSH濃度顯著小於Sham組；於術後6小時，CLP組的iNOS及HMGB1表現量顯著大於Sham組，而血液GSH濃度及GSH/GSSG比例、肝組織GSSG及total GSH濃度顯著小於Sham組；於術後9小時，CLP組的SOD活性及iNOS表現量顯著大於Sham組，而肝組織total GSH濃度顯著小於Sham組；於術後12小時，CLP組的AST及ALT活性、血液GSSG濃度顯著大於Sham組，而WBC數量、ALP活性、血液GSH濃度及GSH/GSSG比例、肝組織total GSH濃度顯著小於Sham組；於術後15小時，CLP組的單核球比例、AST活性、血液GSSG濃度、肝組織NO2濃度顯著大於Sham組，而WBC數量、血液GSH濃度及GSH/GSSG比例顯著小於Sham組。CLP組和CLP+TS組兩者於手術後，相同時間點的比較：於術後0小時，CLP+TS組的肝組織GSH濃度顯著大於CLP組，而肝組織GSSG濃度顯著小於CLP組；於術後3小時，CLP+TS組的SOD活性顯著大於CLP組，而RBC數量及HGB濃度、HMGB1表現量顯著小於CLP組；於術後6小時，CLP+TS組的iNOS及HMGB1表現量顯著小於CLP組；於術後9小時，CLP+TS組的淋巴球比例、血液GSH濃度顯著大於CLP組，嗜中性球比例、ALT活性、肝組織GSSG濃度及GSH/GSSG比例顯著小於CLP組；於術後15小時，CLP+TS組的血液GSH、total GSH濃度及GSH/GSSG比例、肝組織GSSG及NO3濃度顯著大於CLP組，AST及ALT活性、肝組織NO2濃度顯著小於CLP組。於CLP組內，不同時間點的比較發現：RBC數量和HGB濃度於術後3、6小時顯著大於術後0小時，但是術後12、15小時顯著小於術後3小時；嗜中性球比例於術後6、9小時顯著大於術後0、3小時，但是術後12、15小時顯著小於術後9小時；於術後6、9、12、15小時，PLT數量和淋巴球比例顯著小於術後0小時，血液GSH及total GSH濃度顯著低於術後3小時，AST和ALT活性則顯著高於術後0小時；單核球比例於術後15小時顯著大於術後0、3、6小時；ALP活性於術後3、6、9、12小時顯著低於0小時組；SOD活性於術後9小時顯著高於術後0、3、6小時，而術後12、15小時顯著低於術後9小時；血液GSSG濃度於術後3、6、9小時顯著低於術後0小時；血液GSH/GSSG比例於術後12、15小時顯著小於術後3小時；肝組織GSH濃度於術後3、6、12、15小時顯著低於術後0小時；肝組織GSSG濃度及total GSH濃度於術後3、6、9、12、15小時顯著低於術後0小時；肝組織GSH/GSSG比例於術後9、15小時顯著大於術後0、3、6小時；NO2濃度於術後15小時顯著高於術後0、3小時；iNOS表現量於術後3、6、9小時顯著大於術後0小時，術後12、15小時則顯著小於術後3、6、9小時；HMGB1表現量於術後3、6小時顯著大於術後0小時，術後9、12、15小時則顯著小於術後3、6小時。 結論：我們在敗血症小鼠上發現：血液CBC結果異常、抗氧化物的消耗、肝組織的損傷以及氧化傷害增加等現象，補充香椿葉萃取物不僅能夠控制免疫細胞的增加、減少肝組織損傷、減少抗氧化物的消耗以及氧化傷害物質的形成。我們也更進一步發現，香椿葉萃取物之抗氧化效果與HMGB1的調控有關，藉由減少iNOS的表現達到控制氧化傷害物質形成的效果。
並列摘要
Background: Sepsis is a leading cause of mortality in hospitals, leading to inflammatory response and tissue injury due to infection. Previous studies have indicated that sepsis is accompanied by increased oxidative stress. Toona sinensis is a traditional Chinese medicine. This study has demonstrated that Toona sinensis has an antioxidant effect. We used extracts of toona sinensis leaves to investigate the effect of oxidative injury on blood and liver tissue in septic mice. Methods: Male C57BL/6 mice were treated with cecal ligation and puncture (CLP) to induce sepsis. Toona sinensis leaf extracts had been pretreated for 4 weeks before CLP. Mice were separated into 5 groups: the control group, TS group, sham group, CLP group, and CLP+TS group. Blood and liver tissue was sampled 0, 3, 6, 9, 12, and 15 hours after CLP. Blood routine (CBC), liver functions (AST, ALT, ALP), and antioxidants (SOD, GSH) were included in the analysis of blood. Antioxidant (GSH), oxidative injury indicator (NO), oxidative injury related proteins (iNOS, HMGB1) were included in the analysis of the liver tissue. Results: The results showed that the levels of WBC in the blood and the expression of HMGB1 in the liver tissue were significantly decreased in the TS group compared with those of the control group; there was no significant change between the control group and TS group in the other analyses. Compared with the sham group and CLP group at the same time point, levels of blood and liver tissue GSSG in the CLP group were significantly higher, whereas level of blood GSH and GSH/GSSG ratio in the CLP group were significantly lower than the sham group at 0 hr after CLP; levels of RBC, HGB, lymphocyte, blood total GSH, expression of iNOS and HMGB1 in the CLP group were significantly higher, whereas level of neutrophil, liver tissue GSH and total GSH in the CLP group were significantly lower than the sham group at 3 hr after CLP; expression of iNOS and HMGB1 in the CLP group were significantly higher, whereas level of blood GSH and GSH/GSSG ratio, liver tissue GSSG and total GSH in the CLP group were significantly lower than the sham group at 6 hr after CLP; expression of iNOS, activity of SOD in the CLP group were significantly higher, whereas level of liver tissue total GSH in the CLP group were significantly lower than the sham group at 9 hr after CLP; activity of AST and ALT, level of blood GSH in the CLP group were significantly higher, whereas level of WBC, blood GSH and GSH/GSSG ratio, liver tissue total GSH in the CLP group were significantly lower than the sham group at 12 hr after CLP; activity of AST, level of monocyte, blood GSSG, liver tissue NO2 in the CLP group were significantly higher, whereas level of WBC, blood GSH and GSH/GSSG ratio in the CLP group were significantly lower than the sham group at 15 hr after CLP. Compared with the CLP group and CLP+TS group at the same time point, level of liver tissue GSH was significantly higher, whereas level of liver tissue GSSG in the CLP+TS group was significantly lower than the CLP group at 0 hr after CLP; activity of SOD was significantly higher, whereas level of RBC, HGB, expression of HMGB1 in the CLP+TS group was significantly lower than the CLP group at 3 hr after CLP; expression of iNOS and HMGB1 in the CLP+TS group were significantly lower than the CLP group at 6 hr after CLP; level of lymphocyte, blood GSH were significantly higher, whereas activity of ALT, level of neutrophil, liver tissue GSSG and GSH/GSSG in the CLP+TS group was significantly lower than the CLP group at 9 hr after CLP; level of blood GSH, total GSH and GSH/GSSG ratio, liver tissue GSSG and NO3 were significantly higher, whereas activity of AST and ALT, level of liver tissue NO2 in the CLP+TS group was significantly lower than the CLP group at 15 hr after CLP. Comparisons of the time course at different time periods in the CLP group, levels of RBC and HGB at 3, 6hr were higher than 0hr, but 12, 15hr were lower than 3hr; neutrophil ratio at 6, 9hr were higher than 0, 3hr, but 12, 15hr were lower than 9hr; at 6, 9, 12, 15hr, levels of PLT and lymphocyte ratio were lower than 0hr, levels of GSH and total GSH in blood were lower than 3hr, but activity of AST and ALT were higher than 0hr; monocyte ratio at 15hr were higher than 0, 3, 6hr; activity of ALP at 3, 6, 9, 12hr were lower than 0hr; activity of SOD at 9hr were higher than 0, 3, 6hr, but 12, 15hr were lower than 9hr; levels of GSSG in blood at 3, 6, 9hr were lower than 0hr; GSH/GSSG ratio in blood at 12, 15hr were lower than 3hr; levels of GSH in liver tissue at 3, 6, 12, 15hr were lower than 0hr; levels of GSSG and total GSH in liver tissue at 3, 6, 9, 12, 15hr were lower than 0hr; GSH/GSSG ratio in liver tissue at 9, 15hr were lower than 0, 3, 6hr; level of NO2 in liver tissue at 15hr were higher than 0, 3hr; expression of iNOS at 3, 6, 9hr were higher than 0hr, but 12, 15hr were lower than 3, 6, 9hr; expression of HMGB1 at 3, 6hr were higher than 0hr, but 9, 12, 15hr were lower than 3, 6hr. Conclusion: Abnormal CBC results, consumption of antioxidants, liver injury and increased oxidative stress were observed in septic mice. Pretreatment of Toona sinensis leaf extracts could inhibit the increase of immune cells, decrease liver tissue injury and consumption of antioxidants, and prevent the formation of oxidative stress substances in septic mice. The antioxidant ability of Toona sinensis leaf extracts in septic mice was associated with regulation of the HMGB 1 expression by down-regulating iNOS to inhibit oxidative stress.