摘要
目的探讨高乌甲素（LA）对重度烧伤大鼠疼痛和炎症反应的作用及其机制。方法取40只SD大鼠按随机数字表法（下同）分为健康＋生理盐水组、假伤＋生理盐水组、单纯烧伤组、烧伤＋LA组、健康＋LA组，每组8只。单纯烧伤组、烧伤＋LA组大鼠背部及右后肢造成约32％TBSA深Ⅱ度烫伤（以下称烧伤），假伤＋生理盐水组大鼠致假伤。烧伤＋LA组大鼠于伤前2．0h及伤后即刻与伤后24．0、48．0、72．0h分别腹腔注射1g／LLA溶液4mL／kg，健康＋LA组大鼠于同前各时相点腹腔注射相同剂量的LA溶液，健康＋生理盐水组、假伤＋生理盐水组大鼠于同前各时相点腹腔注射生理盐水4mL／kg。于伤前1．5h及伤后12．5、24．5、36．5、48．5、72．5h，观察致伤大鼠疼痛行为并对大鼠行机械缩足反射阈值（PWMT）检测；未致伤2组大鼠于同前各时相点行相同观测。另取32只SD大鼠分为生理盐水组、三硝基苯基（TNP）-ATP组、米诺环素组、磷酸吡哆醛（PPADS）组，每组8只，均同前致烧伤。伤后48．0h，生理盐水组大鼠鞘内注入10μL生理盐水，TNP—ATP组大鼠鞘内注入30nmol／μLTNP-ATP10μL，米诺环素组大鼠鞘内注入5g／L米诺环素10uL，PPADS组大鼠鞘内注入10nmol／μA。PPADS10μL。注射前后0．5h，测量大鼠PWMT。伤后72．5h，取假伤＋生理盐水组、单纯烧伤组、烧伤＋LA组大鼠脊髓背角组织行免疫荧光染色观察P2X。受体和OX42受体共表达情况，行免疫组织化学染色观察P2X。受体表达并计数阳性细胞；取大鼠静脉血采用ELISA法检测血清TNF—α、IL-1β含量。未致伤2组大鼠于同前时相点行相同观测。对数据行单因素方差分析、重复测量方差分析、SNK检验、配对t检验和Bonferroni校正。结果（1）健康＋生理盐水组、假伤＋生理盐水组、健康＋LA组大鼠各时相点未出现活动异常现象。至伤后72．5h，单纯烧伤组大鼠精神状态欠佳，可见背部及右后肢烧伤皮肤红肿、有水疱，出现步态不稳等现象，偶有舔咬、抓挠现象；烧伤＋LA组大鼠舔咬、跛行等行为较单纯烧伤组减少，创面红肿消退较单纯烧伤组快。（2）伤前1．5h，健康＋生理盐水组、假伤＋生理盐水组、单纯烧伤组、烧伤＋LA组、健康＋LA组大鼠PWMT组间总体比较差异无统计学意义（F=0．106，P〉0．05）。单纯烧伤组大鼠伤后各时相点PWMT均明显低于其余4组（P值均小于0．05），烧伤＋LA组大鼠伤后各时相点PWMT均明显低于健康＋生理盐水组、假伤＋生理盐水组和健康＋LA组（P值均小于0．05）。（3）注射前0．5h，生理盐水组、TNP—ATP组、PPADS组、米诺环素组大鼠PWMT相近，分别为15．3±0．8、15．1±1．0、15．3±0．9、15．6±1．1（F=0．343，P〉0．05）。注射后0．5h，生理盐水组和PPADS组大鼠PWMT分别为15．2±1．2和14．8±1．0，明显低于TNP—ATP组和米诺环素组的20．8±1．4和26．3±1．0（P值均小于0．05）。生理盐水组和PPADS组大鼠注射前后PWMT相近（t值分别为0．073和-0．772，P值均大于0．05），TNP—ATP组和米诺环素组大鼠注射后PWMT较注射前升高（t值分别为-10．180和-20．813，P值均小于0．01）。（4）伤后72．5h，健康＋生理盐水组、假伤＋生理盐水组、健康＋LA组大鼠少量小胶质细胞共表达P2X。受体和OX42受体，单纯烧伤组及烧伤＋LA组大鼠大量小胶质细胞出现P2X4受体和OX42受体共表达。伤后72．5h，单纯烧伤组大鼠P2X4受体阳性细胞数较其余4组多（P值均小于0．05），烧伤＋IA组大鼠P2X4受体阳性细胞数明显多于健康＋生理盐水组、假伤＋生理盐水组和健康＋LA组（P值均小于0．05）。（5）伤后72．5h，单纯烧伤组大鼠血清TNF—α和IL-1β含量均明最高于其余4组（P值均小于0．001），烧伤＋LA组大鼠血清TNF—α和IL-1β含量均明显低于健康＋生理盐水组、假伤＋生理盐水组和健康＋LA组（P值均小于0．001）。结论LA对重度烧伤大鼠具有明显镇痛作用，可改善机体过度炎症状态。其作用机制可能与抑制脊髓背角小胶质细胞上P2X4受体表达，减少炎症因子TNF—α、IL—1β释放有关。
Objective To explore the effects of lappaconitine （LA） on pain and inflammatory re- sponse of severely burned rats and the mechanism. Methods Forty SD rats were divided into healthy ＋normal saline group, sham injury ＋normal saline group, pure burn group, burn ＋ LA group, and healthy ＋LA group according to the random number table （the same dividing method below） , with 8 rats in each group. Rats in pure burn and burn ＋LA groups were inflicted with about 32% total body surface area deep partial-thickness scald （hereinafter referred to as burn） on the back and right hind. Rats in sham injury ＋normal saline group were sham injured. Rats in burn ＋LA group were intraperitoneally injected with 1 g/L LA solution in the dosage of 4 mL/kg at 2.0 h before injury and post injury hour （PIH） 0 （immediately） , 24.0, 48.0, and 72.0. Rats in healthy ＋LA group were intraperitoneally injected with LA solution in the same dose at the same time points as above, and rats in healthy ＋normal saline and sham injury ＋normal saline groups were intraperitoneally injected with normal saline in the dose of 4 mL/kg at the same time points as above. At 1.5 h before injury and PIH 12.5, 24.5, 36.5, 48.5, and 72.5, the paw withdrawal mechanical threshold （PWMT） of injured rats was detected, and their pain behaviors were observed. The same observation and detection were conducted in rats without injury in the two groups at the same time points as above. Another 32 SD rats were divided into normal saline group, trinitrophenyl （TNP）-ATP group, minocyline group, pyridoxal-phosphate-6-azophenyl-2＇, 4＇-disulfonic acid （PPADS） group, with 8 rats in each group, and all the rats were inflicted with the same burn injury as above. At PIH 48.0, rats in normal saline group were intrathecally injected with 10 μL normal saline; rats in TNP-ATP group were intrathecally injec- ted with 10 μL TNP-ATP in the concentration of 30 nmol/μL; rats in minocyline group were intrathecally in- jected with 10 μL minocyline in the concentration of 5 g/L; rats in PPADS group were intrathecally injected with 10 μL PPADS in the concentration of 10 nmol/μL. The PWMT of rats was detected at 0.5 h before injection and 0.5 h after. At PIH 72.5, the tissue in the dorsal horn of spinal cord of rats in sham injury ＋normal saline, pure burn, and burn ＋LA groups was harvested to observe the co-expression of P2X4 receptor and OX42 receptor with immunofluorescent staining and to observe the expression of P2X4 receptor and count the positive cells with immunohistochemical staining. The venous blood was harvested for determination of serum content of tumor necrosis factor-alpha （TNF-α） and interleukin-1 beta （ IL-1β） with enzyme-linked immunosorbent assay. The same observation and determination were conducted in rats without injury in the two groups at the same time point as above. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, SNK test, paired t test, and Bonferroni correction. Results （ 1 ） There were no abnormal activity in rats of healthy ＋normal saline, sham injury ＋normal saline, healthy ＋LA groups at all time points. Until PIH 72.5, rats in pure burn group were in poor mental state; red and swollen manifestation and blister were observed in burn wounds on the back and right hind; imbalance in gait, lick, bite, and scratch were observed occasionally. Fewer behaviors such as lick, bite, and limp were observed in rats in burn ＋LA group than in pure burn group, and the red and swollen manifestation in wounds of rats in burn ＋LA group dissipated faster than that in pure burn group. （2） At 1.5 h before injury, there were no significant differences in the PWMT values of rats in healthy ＋normal saline, sham injury ＋normal saline, pure burn, burn＋A, and healthy ＋A groups （ F =0. 106, P 〉0.05）. PWMT values of rats in pure burn group were significantly lower than those in the other 4 groups at all post injury time points （with P val- ues below 0.05）. PWMT values of rats in burn ＋LA group were significantly lower than those in heahhy ＋normal saline, sham injury ＋normal saline, and healthy ＋LA groups at all post injury time points （ with P values below 0.05）. （3） At 0.5 h before injection, PWMT values of rats in normal saline, TNP-ATP, PPADS, and minocyline groups were close, respectively 15.3±0.8, 15.1±1.0, 15.3 ±0.9, and 15.6±1.1 （ F =0. 343, P 〉0.05）. At 0.5 h after injection, PWMT values of rats in normal saline group and PPADS group were respectively 15.2 ±1.2 and 14.8±1.0, which were significantly lower than 20.8±1.4 and 26.3 ±1.0 in TNP-ATP group and minocyline group respectively （ with P values below 0.05）. PWMT values of rats in normal saline and PPADS groups were similar before and after injection （ with t values re- spectively 0. 073 and - 0. 772, P values above 0.05） , while those of rats in TNP-ATP and minocyline groups were higher after injection than before injection （with t values respectively - 10. 180 and -20. 813, P values below 0.01 ）. （4） At PIH 72.5, co-expression of P2X4 receptor and OX42 receptor was observed in a few microglias of rats in healthy ＋normal saline, sham injury ＋normal saline, and healthy ＋LA groups,while co-expression of P2X4 receptor and OX42 receptor was observed in a large number of microglias of rats in pure burn and burn ＋LA groups. At PIH 72.5, more P2X4 receptor positive cells were observed in rats in pure burn group than in the other 4 groups （with P values below O. 05 ） , and more P2X4 receptor positive cells were observed in rats in burn ＋LA group than in healthy ＋normal saline, sham injury ＋normal saline, and healthy ＋LA groups （with P values below 0, 05）. （5） At Pill 72.5, the serum content of TNF-αand IL-1β of rats in pure burn group was significantly higher than that in the other 4 groups （with P values below 0. 001 ）. The serum content of TNF-α＋and IL-1β of rats in burn ＋LA group was significantly lower than that in healthy ＋normal saline, sham injury ＋normal saline, and healthy ＋LA groups （ with P values below 0. 001 ）. Conclusions LA has significant analgesic effects on severely burned rats, and it can ameliorate the excessive inflammational situation. The mechanism may he related to its inhibition of expression of P2X4 receptor in mieroglias in the dorsal horn of spinal cord and reduction in the release of inflammatory factors TNF-α and 1L-1β.
作者
杨成兰
魏在荣
张天华
曾雪琴
吴必华
Yang Chenglan Wei Zairong Zhang Tianhua Zeng Xueqin Wu Bihua(Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical College, Zunyi 563003, Chin)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2017年第6期374-380,共7页
Chinese Journal of Burns
基金
贵州省科教青年英才培养工程项目（黔省专合字[2012]191号）
汇川区科技计划项目
