Effects of lappaconitine on pain and inflammatory response of severely burned rats and the mechanism

Objective To explore the effects of lappaconitine (LA) on pain and inflammatory response 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 injected with 10 μL TNP-ATP in the concentration of 30 nmol/μL; rats in minocyline group were intrathecally injected 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+ LA, and healthy+ LA 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 values below 0.05). PWMT values of rats in burn+ LA group were significantly lower than those in healthy+ 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 respectively 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 0.05), 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 PIH 72.5, the serum content of TNF-α and 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 be related to its inhibition of expression of P2X₄ receptor in microglias in the dorsal horn of spinal cord and reduction in the release of inflammatory factors TNF-α and IL-1β.