不同剂量乌司他丁对脓毒症大鼠炎症因子及细胞免疫的调节作用

Effects of different doses of ulinastatin on inflammatory factors and cellular immunity in septic rats

目的观察乌司他丁对脓毒症大鼠炎症因子及细胞免疫的调节作用。方法按随机数字表法将290只雄性SD大鼠分为假手术组(A组) 15只,模型组(B组)、治疗组(C组)、实验1组(D组)、实验2组(E组)、实验3组(F组)各55只,B、C、D、E、F组采用盲肠结扎打孔制备脓毒症模型,A组只开腹、关腹。建模成功后剔除死亡大鼠,A组及B组术后皮下注射生理盐水;C组在B组的基础上应用左氧氟沙星;D、E、F组分别在C组基础上应用乌司他丁10万、25万、50万U/kg。各组大鼠观察至6 h(T1)、24 h(T2)、48 h(T3)时,分别取当时存活大鼠10只处死后取血,用ELISA法检测血清IL-6、TNF-α、IL-10,采用流式细胞仪检测T淋巴细胞亚群。结果与B组比较,T3时C、D、E、F组IL-6、TNF-α、IL-10降低,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)比值升高[IL-6(pg/m L):256.72±32.28 vs. 226.32±29.10(C) vs. 198.82±25.99(D) vs. 173.55±21.80(E) vs.170.82±23.70(F);TNF-α(pg/m L):105.96±16.36 vs. 89.93±15.87(C) vs. 75.25±13.48(D)vs. 62.88±10.25(E) vs. 61.43±10.46(F);IL-10(pg/m L):36.48±6.30 vs. 29.48±5.97 (C) vs.24.22±4.06(D) vs. 19.58±3.04(E) vs. 18.78±3.18(F);CD3^(+)(%):39.77±4.45 vs. 44.28±3.99(C) vs. 48.10±4.79(D) vs. 53.88±5.09(E) vs. 52.33±5.39(F);CD4^(+)(%):28.65±3.26 vs.32.79±3.23(C) vs. 36.19±2.74(D) vs. 39.04±2.30 (E) vs. 39.42±3.17 (F);CD4^(+)/CD8^(+)(%):48.19±6.07 vs. 55.02±7.43(C) vs. 62.80±5.89(D) vs. 88.48±9.42(E) vs. 87.56±8.79(F);均P <0.05];与B组比较,T2时E、F组IL-6、TNF-α、IL-10降低,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)比值升高[IL-6(pg/m L):268.84±30.50 vs. 228.58±33.47 (E) vs. 229.27±32.28 (F);TNF-α(pg/m L):114.11±16.67 vs. 96.64±16.66(E) vs. 95.56±15.44(F);IL-10(pg/m L):19.16±4.73 vs. 15.33±2.95(E) vs. 15.18±2.51 (F);CD3^(+)(%):44.36±4.21 vs. 49.50±4.47(E) vs.48.31±4.73(F);CD4^(+)(%):33.24±3.89 vs. 37.63±2.95(E) vs. 36.76±4.01(F);CD4^(+)/CD8^(+)(%):81.34±8.69 vs. 92.90±6.35(E) vs. 93.44±7.48(F);均P <0.05];与C组比较,T3时D、E、F组IL-6、TNF-α、IL-10降低,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)比值升高[IL-6(pg/m L):226.32±29.10 vs. 198.82±25.99(D) vs. 173.55±21.80(E) vs. 170.82±23.70(F);TNF-α(pg/m L):89.93±15.87 vs. 75.25±13.48(D) vs. 62.88±10.25 (E) vs. 61.43±10.46 (F);IL-10 (pg/m L):29.48±5.97 vs. 24.22±4.06(D) vs. 19.58±3.04 (E) vs. 18.78±3.18 (F);CD3^(+)(%):44.28±3.99 vs.48.10±4.79(D) vs. 53.88±5.09 (E) vs. 52.33±5.39 (F);CD4^(+)(%):32.79±3.23 vs. 36.19±2.74(D) vs. 39.04±2.30 (E) vs. 39.42±3.17 (F);CD4^(+)/CD8^(+)(%):55.02±7.43 vs. 62.80±5.89(D) vs. 88.48±9.42(E) vs. 87.56±8.79(F);均P <0.05]。与D组比较,T3时E、F组IL-6、TNF-α、IL-10降低,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)比值升高[IL-6(pg/m L):198.82±25.99 vs.173.55±21.80(E) vs. 170.82±23.70(F);TNF-α(pg/m L):75.25±13.48 vs. 62.88±10.25 (E)vs. 61.43±10.46 (F);IL-10 (pg/m L):24.22±4.06 vs. 19.58±3.04 (E) vs. 18.78±3.18 (F);CD3^(+)(%):48.10±4.79 vs. 53.88±5.09(E) vs. 52.33±5.39(F);CD4^(+)(%):36.19±2.74 vs.39.04±2.30 (E) vs. 39.42±3.17 (F);CD4^(+)/CD8^(+)(%):62.80±5.89 vs. 88.48±9.42(E) vs.87.56±8.79(F);均P <0.05]。结论乌司他丁可减轻脓毒症大鼠的炎症反应,改善脓毒症大鼠的细胞免疫功能,应用25万U/kg剂量较为合理。

To observe the effect of ulinastatin on inflammatory factors and cellular immunity in septic rats. Methods 290 male SD rats were randomly divided into sham operation group( group A)and model group( group B), treatment group( group C), experimental group 1( group D),experimental group 2( group E),experimental group 3( group F),the rats in group A were given laparotomy and abdominal closure,and other groups were used to prepare sepsis models by cecal ligation and puncture. The rats in group A and group B were subcutaneously injected with normal saline. The rats in group C were injected with levofloxacin and normal saline. The rats in group D,E and F were injected with ulinastatin 100 000 U/kg,250 000 U/kg and 500 000 U/kg besides levofloxacin and normal saline. At 6 h( T1),24 h( T2) and 48 h( T3),10 surviving rats at that time were sacrificed and the blood was collected. The serum IL-6,TNF-α and IL-10 levels were detected by ELISA,and the T lymphocyte subsets were detected by flow cytometry. Results Compared with group B,IL-6,TNF-α and IL-10 in groups C,D,E and F decreased,while CD3^(+),CD4^(+),CD4^(+)/CD8^(+)ratio increased at T3 [IL-6( pg/m L) : 256. 72 ± 32. 28 vs. 226. 32 ± 29. 10( C) vs. 198. 82 ± 25. 99( D) vs.173. 55 ± 21. 80( E) vs. 170. 82 ± 23. 70( F);TNF-α( pg/m L) : 105. 96 ± 16. 36 vs. 89. 93 ± 15. 87( C)vs. 75. 25 ± 13. 48( D) vs. 62. 88 ± 10. 25( E) vs. 61. 43 ± 10. 46( F);IL-10( pg/m L) : 36. 48 ± 6. 30 vs. 29. 48 ± 5. 97( C) vs. 24. 22 ± 4. 06( D) vs. 19. 58 ± 3. 04( E) vs. 18. 78 ± 3. 18( F);CD3^(+)( %) :39. 77 ± 4. 45 vs. 44. 28 ± 3. 99( C) vs. 48. 10 ± 4. 79( D) vs. 53. 88 ± 5. 09( E) vs. 52. 33 ± 5. 39( F);CD4^(+)( %) : 28. 65 ± 3. 26 vs. 32. 79 ± 3. 23( C) vs. 36. 19 ± 2. 74( D) vs. 39. 04 ± 2. 30( E) vs. 39. 42 ±3. 17( F);CD4^(+)/CD8^(+)( %) : 48. 19 ± 6. 07 vs. 55. 02 ± 7. 43( C) vs. 62. 80 ± 5. 89( D) vs. 88. 48 ±9. 42( E) vs. 87. 56 ± 8. 79( F);all P < 0. 05]. Compared with group B,IL-6,TNF-α and IL-10 decreased,CD3^(+),CD4^(+),and CD4^(+)/CD8^(+)ratio increased in groups E and F at T2 [IL-6( pg/m L) :268. 84 ± 30. 50 vs. 228. 58 ± 33. 47( E) vs. 229. 27 ± 32. 28( F);TNF-α( pg/m L) : 114. 11 ± 16. 67 vs. 96. 64 ± 16. 66( E) vs. 95. 56 ± 15. 44( F);IL-10( pg/m L) : 19. 16 ± 4. 73 vs. 15. 33 ± 2. 95( E) vs.15. 18 ± 2. 51( F);CD3^(+)( %) : 44. 36 ± 4. 21 vs. 49. 50 ± 4. 47( E) vs. 48. 31 ± 4. 73( F);CD4^(+)( %) :33. 24 ± 3. 89 vs. 37. 63 ± 2. 95( E) vs. 36. 76 ± 4. 01( F);CD4^(+)/CD8^(+)( %) : 81. 34 ± 8. 69 vs. 92. 90 ±6. 35( E) vs. 93. 44 ± 7. 48( F);all P < 0. 05]. Compared with group C,IL-6,TNF-α and IL-10 decreased,CD3^(+),CD4^(+),and CD4^(+)/CD8^(+)ratio increased in groups D,E and F at T3[IL-6( pg/m L) :226. 32 ± 29. 10 vs. 198. 82 ± 25. 99( D) vs. 173. 55 ± 21. 80( E) vs. 170. 82 ± 23. 70( F);TNF-α( pg/m L) : 89. 93 ± 15. 87 vs. 75. 25 ± 13. 48( D) vs. 62. 88 ± 10. 25( E) vs. 61. 43 ± 10. 46( F);IL-10( pg/m L) : 29. 48 ± 5. 97 vs. 24. 22 ± 4. 06( D) vs. 19. 58 ± 3. 04( E) vs. 18. 78 ± 3. 18( F);CD3^(+)( %) :44. 28 ± 3. 99 vs. 48. 10 ± 4. 79( D) vs. 53. 88 ± 5. 09( E) vs. 52. 33 ± 5. 39( F);CD4^(+)( %) : 32. 79 ±3. 23 vs. 36. 19 ± 2. 74( D) vs. 39. 04 ± 2. 30( E) vs. 39. 42 ± 3. 17( F);CD4^(+)/CD8^(+)( %) : 55. 02 ±7. 43 vs. 62. 80 ± 5. 89( D) vs. 88. 48 ± 9. 42( E) vs. 87. 56 ± 8. 79( F);all P < 0. 05]. Compared with group D,IL-6,TNF-α and IL-10 decreased,CD3^(+),CD4^(+),CD4^(+)/CD8^(+)ratio increased in group E and F at T3[IL-6( pg/m L) : 198. 82 ± 25. 99 vs. 173. 55 ± 21. 80( E) vs. 170. 82 ± 23. 70( F);TNF-α( pg/m L) : 75. 25 ± 13. 48 vs. 62. 88 ± 10. 25( E) vs. 61. 43 ± 10. 46( F);IL-10( pg/m L) :24. 22 ± 4. 06 vs. 19. 58 ± 3. 04( E) vs. 18. 78 ± 3. 18( F);CD3^(+)( %) : 48. 10 ± 4. 79 vs. 53. 88 ± 5. 09( E) vs. 52. 33 ± 5. 39( F);CD4^(+)( %) : 36. 19 ± 2. 74 vs. 39. 04 ± 2. 30( E) vs. 39. 42 ± 3. 17( F);CD4^(+)/CD8^(+)( %) : 62. 80 ± 5. 89 vs. 88. 48 ± 9. 42( E) vs. 87. 56 ± 8. 79( F);all P < 0. 05].Conclusions Ulinastatin can reduce the inflammatory response and improve the cellular immune function of septic rats,and the application of 250 000 U/kg dose is reasonable to achieve the ideal curative effect.