温阳振衰颗粒对脓毒症大鼠炎症反应中SIRS/CARS平衡调节的影响

Regulation effect of Wenyang Zhenshuai Granule on SIRS/CARS balancein inflammatory reaction of rats with sepsis

目的探讨温阳振衰颗粒对脓毒症大鼠炎症反应的调节作用.方法将56只SD大鼠按随机数宇表法分为假手术组(n=8)、模型组(n=24)、中药治疗组(n=24).采用腹腔注射脂多糖(LPS)5 mg/kg的方法制备脓毒症大鼠模型;假手术组给予等量生理盐水.中药治疗组于制模后即刻灌胃温阳振衰颗粒原药(250 g/L)2 g·kg^-1·d^-1;模型组灌胃等量生理盐水;此后存活动物每12 h给药(或生理盐水);假手术组不予处理.各组分别于制模后24、48、72 h取8只大鼠,经眼眶采血后处死,采用酶联免疫吸附试验(ELISA)测定血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-10)水平;采用聚合酶链反应(PCR)检测肠组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子-κB(NF-κB)的mRNA表达.结果与假手术组比较,模型组制模后24 h血清中TNF-α、IL-6水平即显著升高[TNF-α(ng/L):32.03±2.01比13.70±1.06,IL-6(ng/L):50.36±2.66比27.90±1.08],IL-10水平即显著下降(ng/L:80.96±4.90比90.82±4.85),肠组织TLR4、MyD88、NF-κB的mRNA表达即显著升高(TLR4/β-actin:0.39±0.06比0.21±0.04,MyD88/β-actin:0.38±0.04比0.18±0.02,NF-κB/β-actin:0.40±0.04比0.20±0.03),差异均有统计学意义(均P<0.05);巨随着时间延长,血清TNF-α、IL-6水平及肠组织TLR4、MyD88、NF-κB的mRNA表达逐渐升高,血清IL-10水平逐渐下降.与模型组比较,中药治疗组制模后24 h血清TNF-α、IL-6水平及肠组织TLR4、MyD88、NF-κB的mRNA表达即显著下降[TNF-α(ng/L):31.45±2.40比32.03±2.01,IL-6(ng/L):42.84±3.12比50.36±2.66,TLR4/β-actin:0.32±0.05比0.39±0.06,MyD88/β-actin:0.30±0.05比0.38±0.04,NF-κB/β-actin:0.33±0.03比0.40±0.04],制模后48 h血清IL-10水平即显著升高(ng/L:73.09±3.26比62.93±4.98),差异均有统计学意义(均P<0.05);巨随时间延长,血清TNF-α、IL-6水平及肠组织TLR4、MyD88、NF-κB的mRNA逐渐下降.结论温阳振衰颗粒可能通过早期激发抗炎因子平衡炎症反应,以及抑制TLR4、MyD88、NF-κB的mRNA表达从而使该通路下游炎症因子活化减弱,避免炎症"瀑布效应",最终使脓毒症大鼠全身炎症反应综合征/代偿性抗炎反应综合征(SIRS/CARS)两者达到平衡.

Objective To observed the regulation effect of Wenyang Zhenshuai Granule on inflammatory reaction of rats with sepsis.Methods Fifty-six Sprague-Dawley(SD)rats were divided into sham operation group(Sham group,n=8),model group(n=24),and Chinese medicine treatment group(n=24)according to random number table method.Rats modelling with sepsis were developed by intraperitoneal injection of lipopolysaccharide(LPS,5 mg/kg).Rats in Sham group were given the same amount of normal saline by intraperitoneal injection.After modeling,rats in the Chinese medicine treatment group took Wenyang Zhenshuai Granule orally(250 g/L)2 g·kg^-1·d^-1,meanwhile rats in the model group were given equal amount of normal saline by intragastric administration.After that,the surviving animals were given medicine(or normal saline)every 12 hours;the sham group was not treated.Eight rats in each group were taken at 24,48 and 72 hours after the preparation of the membrane,and then blood was collected from the orbit and sacrificed,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL-6,IL-10)in serum were determined by enzymelinked immunosorbent assay(ELISA);and the mRNA expressions of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88)and nuclear transcription factor-κB(NF-κB)in intestinal tissue were detected by polymerase chain reaction(PCR).Results Compared with the Sham group,the levels of TNF-αand IL-6 in the serum of the model group significantly increased 24 hours after membrane preparation[TNF-α(ng/L):32.03±2.01 vs.13.70±1.06,IL-6(ng/L):50.36±2.66 vs.27.90±1.08],serum IL-10 level significantly decreased(ng/L:80.96±4.90 vs.90.82±4.85),the mRNA expressions of TLR4,MyD88 and NF-κB in intestinal tissue significantly increased(TLR4/β-actin:0.39±0.06 vs.0.21±0.04,MyD88/β-actin:0.38±0.04 vs.0.18±0.02,NF-κB/β-actin:0.40±0.04 vs.0.20±0.03),with statistically significant differences(all P<0.05);with the increase of time,the levels of TNF-αand IL-6 in serum,the mRNA expressions of TLR4,MyD88 and NF-κB in intestinal tissue increased gradually,and serum IL-10 level decreased gradually.Compared with the model group,the levels of TNF-αand IL-6 in serum and the mRNA expressions of TLR4,MyD88 and NF-κB in intestinal tissue in the Chinese medicine treatment group decreased significantly 24 hours after model preparation[TNF-α(ng/L):31.45±2.40 vs.32.03±2.01,IL-6(ng/L):42.84±3.12 vs.50.36±2.66,TLR4/β-actin:0.32±0.05 vs.0.39±0.06,MyD88/β-actin:0.30±0.05 vs.0.38±0.04,NF-κB/β-actin:0.33±0.03 vs.0.40±0.04],the serum IL-10 level increased significantly 48 hours after preparation(ng/L:73.09±3.26 vs.62.93±4.98),with statistically significant differences(all P<0.05),With the increase of time,the levels of TNF-αand IL-6 in serum,the mRNA expressions of TLR4,MyD88 and NF-κB in intestinal tissue decreased gradually.Conclusions Wenyang Zhenshuai Granule reduced the inflammatory cytokines expression and stimulated the anti-inflammatory cytokines of rats with sepsis induced by CLP so as to alleviate or avoid SIRS.Its mechanism may be related to motivating balance among anti-inflammatory cytokines in the early stage,inhibiting TLR4,MyD88 and NF-κB mRNA expressions,and weaken activation of the inflammatory cytokines in the pathway downstream which avoided waterfall effect in inflammation and balanced the systemic inflammatory response syndrome/compensatory anti-inflammatory response syndrome(SIRS/CARS).