肝醣分解及自然免疫指標在致死性或次致死性敗血症的實驗動物模式的角色

Role of Glycogenolytic and Innate Immunological Markers during Lethal and Sublethal Sepsis in Experimental Animal Model

林 吉吉(Che Lin)

指導教授 ： 林清淵

長榮大學/健康科學學院/醫學研究所/碩士(2007年)

https://doi.org/10.6833/CJCU.2007.00049

摘要

細菌敗血症會致命的機轉，假說很多，聚焦在一個假說:”在致命低血糖， 能否在適當時間、適當反應，決定是否存活”; MyD88獨立路徑可能使血糖利用增加，藉由MyD88獨立路徑連鎖反應後線產物的測量， 和血糖相關影響因子的分析(血糖、血清胰島素值、肝臟葡萄糖-6-磷酸酶、肝臟葡萄糖6磷酸轉送酶、肝臟干擾素-beta)，找出和細菌敗血症致命低血糖的相關性因子。
試驗致病菌大腸桿菌ATCC259222的致死劑量(LD， 注射後72小時內小鼠100% 致死的最低劑量); 四十八隻BALB/c 6週齡雄性小鼠， 分為三組(控制組、近致死組、致死組)， 除了控制組， 近致死組(0.1LD， 給予1/10倍致死劑量)及致死組(10LD， 給予10倍致死劑量)， 腹腔內注射致病菌大腸桿菌ATCC25922， 控制組注射等體積的LB培養液。結果顯示:在次致死組血清胰島素值會逐漸的增加(第6小時、第3天、第7天)次致死組和致死組比較時， 次致死組血清胰島素值在第6小時較高， 具統計學上的差異(P<0.05);在第6小時， 次致死組(0.1LD)和致死組(10LD)都會降低肝臟葡萄糖-6-磷酸酶活性(和控制組比較的百分比， 次致死組51±32% vs. 致死組72±23 % ， P> 0.05 ); 然而，在次致死組肝臟葡萄糖-6-磷酸酶活性會隨著血糖反彈增加(第3天、第7天)， 次致死組在第三天增加最多肝臟葡萄糖-6-磷酸酶活性 (和控制組比較的百分比，控制組，第6小時， 100±8%;次致死組，第6小時， 51±9%; 次致死組， 第3天， 108±6%; 次致死組，第7天， 103±6%;致死組，第6小時， 72±9%); 次致死組和致死組比較時，在第6小時次致死組肝臟葡萄糖6磷酸轉送酶訊息核醣核酸較低，(和控制組比較的百分比， 次致死組32±4% vs. 致死組144±32 % ，具統計學上的差異， P<0.05);肝臟葡萄糖6磷酸轉送酶訊息核醣核酸次致死組在第七天增加最多(和控制組比較的百分比，控制組，第6小時， 100±4%;次致死組，第6小時， 32±4%; 次致死組， 第3天， 231±17%; 次致死組，第7天， 412±30%;致死組，第6小時， 144±32 %);肝臟葡萄糖-6-磷酸酶及肝臟葡萄糖6磷酸轉送酶， 在三組間有統計差異， 和敗血症動物的死亡有關係。在注射致病菌6小時， 肝臟 TRAM (MyD88獨立路徑上游)，近致死組肝臟TRAM表現量比致死組要高 (368±33 vs. 29±6 %和控制組比較， P<0.05); MyD88獨立路徑中游的IRF-3 和干擾素-beta在組織免疫化學反應有相近的反應; 在肝臟西方點墨反應結果，MyD88獨立路徑中游的IRF-3以及下游的晚期NF-κB也有相似的反應。
壹、中文摘要
細菌敗血症會致命的機轉，假說很多，聚焦在一個假說:”在致命低血糖， 能否在適當時間、適當反應，決定是否存活”; MyD88獨立路徑可能使血糖利用增加，藉由MyD88獨立路徑連鎖反應後線產物的測量， 和血糖相關影響因子的分析(血糖、血清胰島素值、肝臟葡萄糖-6-磷酸酶、肝臟葡萄糖6磷酸轉送酶、肝臟干擾素-beta)，找出和細菌敗血症致命低血糖的相關性因子。
總結，除了傳統的MyD88非獨立路徑，對於致命的敗血症低血糖，能否即時啟動MyD88獨立路徑、TRAM訊息核醣核酸適當表現、晚期NF-κB之磷酸化、IRF-3蛋白增加、干擾素-beta蛋白增加、使血糖利用增加，可能是細菌敗血症存活的關鍵。

關鍵字

胰島素 ； 葡萄糖-6-磷酸酶葡萄糖6磷酸轉送酶MyD88 ； 干擾素-beta

並列摘要

We focused on the hypothesis that rebound hypoglycemia with activating MyD88-independent pathway are required for the survival of lethal hypoglycemia in sepsis. Forty-eight mice (BALB/c) were divided into three groups (control, sublethal, and lethal group).
The results showed that at 6 hours sublethal dose E. coli (0.1LD) decreased blood glucose as the same as lethal dose E. coli (10LD). The blood insulin concentrations were significantly higher at 6 hours (P<0.05) in the sublethal group compared with those in the lethal group. Liver G6Pase activity levels rebounded with blood glucose at 3 days and 7 days in the sublethal group. The sublethal group induced the greatest fold increase of liver G6Pase activity at 3 days. Liver G6PT mRNA measured by realtime PCR revealed liver G6PT levels were lower at 6 hours in the sublethal group compared with those in the lethal group. The sublethal group induced the greatest fold increase of liver G6PT mRNA at 7 days. Liver TRAM mRNA measured by realtime PCR revealed liver TRAM levels were higher at 6 hours in the sublethal group compared with those in the lethal group. The sublethal group induced the greatest fold increase of liver G6PT mRNA at 3 days. These results indicated that the lethal group reduced TRAM production and subsequent expression of IRF-3 and IFN-beta proteins in liver IHC. The increase in NF-kappaB protein was accounted for by western blot in the sublethal group and compared with those in the lethal group at 6 hours. The level of NF-kappaB protein increased 6 hours through 7days progressively in the sublethal group. The increase in IRF-3 protein was accounted for by western blot in the sublethal group and compared with those in the lethal group. The level of IRF-3 protein increased progressively from 6 hours to 7 days in the sublethal group. The levels of IRF-3 in lethal group were lower than those of control group. The sublethal group induced the greatest increase of liver IFN-beta protein at 3 days. This data suggests that lethal dose E. coli reduces activation of IFN-beta. Interestingly, blood insulin decrease was synchronic with IFN-beta decrease.
Finally, compared with a sublethal dose of E. coli ATCC 25922, sepsis induced by a lethal dose causes blood insulin decrease, liver G6PT mRNA expression increase, liver TRAM mRNA expression and protein decrease, liver NF-kappaB protein decrease, liver IRF-3 protein decrease, and liver IFN-beta protein decrease.

並列關鍵字

insulin ； glucose 6 phosphatase ； glucose 6 phosphate transporter ； myeloid differentiation factor 88 ； interferon beta

參考文獻

1. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS. Role of transcriptional activation of I kappa B alpha in medication of immunosuppresion by glucocorticoids. Science. 1995;13:270:283-6.

3. Maitra SR, Homan CS, Pan W, Geller ER, Henry MC, Thode HC. Renal gluconeogenesis and blood flow during endotoxic shock. Acad Emerg Med. 1996;3:1006-10.

4. Losek JD. Hypoglycemia and the ABC'S (sugar) of pediatric resuscitation. Ann Emerg Med. 2000;35:43-6.

5. Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest. 2006;116:3015-25.

6. Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T, et al. TRAM is specifically involved in the Toll-like receptor 4-mediated Myd88-independent signaling pathway. Nat Immunol. 2003;4:1144-50.