Immunosuppresive, antioxidative, anti-inflammatory and analgesic effect of Xiahuoluo pills / 中国组织工程研究

ABSTRACT

BACKGROUND: Xiaohuoluo pill can expel pathogenic wind, remove dampness and activate collaterals. It is used for treatment of Bi-syndrome due to wind-cold-dampness, pain and numbness in limbs.OBJECTIVE: To observe the pharmacological effect of Xiaohuoluo pills on secondary immune response, specific immunity (including cellular immunity and humoral immunity), non-specific immunity [including complement 3(C3), mononuclear phagocyte system (MPS) and red blood cell (RBC)adhesion function] and free radical injury as well as pain and many other inflammations in mice.DESIGN: A randomized controlled stratified trial.SETTING: Guangzhou Institute of Traditional Chinese Medicine and Chinese Materia Medica; Department of Pharmacy, Guangzhou Hospital of Traditional Chinese Medicine.MATERIALS Totally 628 NIH and ICR mice of 6 to 8 weeks were involved in this trial. Xiaohuoluo pills (components Dannanxing, Zhichuanwu, Zhicaowu, Dilong, Ruxiang and so on; Chenli Pharmaceutical Factory,Guangzhou; Brach No. 19980612) were used in this trial. Rabbit antimouse immunoglobulin G (IgG) and C3 antiserum reagent kit (Guangzhou Institute of Medicine and Health) and reagent kit for measuring the antioxidizing activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) (Jiancheng Institute of Bioengineering, Nanjing) were used.METHODS: This trial was carried out in the Guangzhou Institute of Traditional Chinese Medicine and Chinese Materia Medica; Department of Pharmacy, Guangzhou Institute of Medicine and Health during September 1998 to December 1999. ① To observe the suppressive effect of Xiaohuoluo pills on cock red blood cell (CRBC)-induced secondary immune response Eight-four ICR mice, male and female in half, were selected.Twenty of 84 mice served as blank controls; The other 64 mice were intraperitoneally injected with cyclophos-phamide (CY) of 0.2 g/kgonce. On the 4th and 12th days, CRBC was intraperitoneally injected into the mice twice to induce immunoenhancing pathological models to form secondary immune response. Mice served as blank controls were intraperitoneally injected with the same volume of normal saline; The immunoenhanced mice were assigned into 3 groups by a lot CY group (n=20, CY, 40 mg/kg, intragastric administration, I.g.), Xiaohuoluo pills (n=21, Xiaohuoluo pills suspension, 5.54 g/kg, I.g.) and model group (n=20, distilled water, the same volume as other groups, I.g.); once a day within 7 successive days. 19 days later, the levels of serum IgG and C3 were measured with single immunodiffusion method, and the level of circulating immune compound (CIC) was measured with polyethylene glycol precipitation method. ② To observe the suppressive effect of Xiaohuoluo pills on delayed type hypersensitivity (DTH) Fifty-four ICR mice, male and female in half, were selected. On the 1st day, the mice were sensitized by subcutaneous injection of 10 g/L 2,4-dinitrofluorobenzen e (DNFB) of 50 μL for each. On the 4th day, the sensitized mice were assigned into 3 groups by a lot Prednisone group (n=18, prednisone, 0.01 g/kg, I.g.), Xiaohuoluo pills (n=18, Xiaohuoluo pills suspension, 5.54 g/kg, I.g.), model group (n=18, distilled water, the same volume as other groups, I.g.), all once a day within 7 successive days. 11 days later, 10 g/L DNFB of 25μL was spread on the right ear of each mouse in each group. The swelling degree was calculated 24 hours later (The mass difference between right ear and left ear). ③ To observe the suppressive effect of Xiaohuoluo pills on immune adhesion function of RBC of mouse Thirty-six NIH mice, male and female in half, were selected and assigned into 3 groups by a lot CY group (n=12, CY, 20 mg/kg,I.g.), Xiaohuoluo pills (n=12, Xiaohuoluo pills suspension, 5.54 g/kg, I.g.)and blank control group (n=12, distilled water, the same volume as other groups, I.g.), once a day within 7 successive days. 7 days later, blood was taken from the orbit of mice for calculating the rosette rate of RBC-C3b receptor and the rosette rate of RBC immune compound. ④ To observe the suppressive effect o20 Mg/kg, I.g.),Xiaohuoluo pills group (Xiaohuoluo pills suspension, 5.54 g/kg, I.g.) , once a day within 7 successive days; IgM-type hemolytic concentration (HC50)was measured at 2 hours after the last administration on the 7th day [ (Sample absorption / Absorption at HC50 of CRBC) ×diluted time]. The levels of serum C3 and MDA and the activity of SOD were measured according to the method from corresponding reagent kit. ⑥ To observe the suppressive effect of Xiaohuoluo pills on agar granulation tissue hyperplasia in miceFifty-nine NIH mice were selected and given subcutaneous injection of 20 g/L agar of 0.5 mL for each. 24 hours later, the mice were assigned into 3 groups by a lot diclofenac group (diclofenac, 10 mg/kg,I.g..), Xiaohuoluo pills group (Xiaohuoluo pills suspension, 5.54 g/kg, I.g.) and model group (distilled water, the same volume as other groups, I.g.), once a day within 7 successive days; On the 8th day, the mice were sacrificed. The hyperplasiainhibiting effect was presented in the form of the mass of agar granulation tissue in one kilogram body mass ⑦ To observe the suppressive effect of Xiaohuoluo pills on acetic distortion reaction Sixty-three NIH mice were se lected and assigned into 3 groups by a lot diclofenac group (diclofenac,50 mg/kg, I.g.), Xiaohuoluo pills group (Xiaohuoluo pills suspension, 5.54 g/kg,I.g.) and model group (distilled water, the same volume of other groups, I.g.),once a day within 2 successive days. At 2 hours after the last administration, the mice were given intraperitoneal injection of 0.1 mol/L acetic acid of 0.2 mL for each one. The times of distortion of mice within 20 minutes were counted. ⑧ To observe the effect of Xiaohuoluo pills on the acute exudative inflammation evoked by dimethylbenzene, croton oil and carrageenan, and the level of prostaglandin E in the inflammatory exudatesTotally 219 NIH mice were selected and assigned into 3 groups by a lotdiclofenac group (diclofenac, 50 mg/kg, I.g.), Xiaohuoluo pills group (Xiaohuoluo pills suspension, 5.54 g/kg, I.g.) and model group (distilled water, the same volume of other groups, I.g.) once a day within 2 successive days. At 2 hours after the last administration, dimethylbenzene of 25 μL was spread on the right ear for 20 minutes, or croton oil of 25 μL was also spread on the right ear, 4 hours later, the swelling of right ear was calculated (mass of right ear-mass of left ear). 10 g/L carrageenan of 20 μL was subcutaneously injected into the right foot, 3 hours later, the swelling degree was calculated (The difference of right foot and left foot); and the level of prostaglandin E in the inflammatory exudates was measured. ⑨ t test(t' test for heteroscedasticity) was used for comparing the difference in measurement data among groups.MAIN OUTCOME MEASURES: Pharmacological effect of Xiaohuoluo pills on secondary immune response, specific immunity, non-specific immunity and free radical injury as well as pain and many other inflammations in mice.RESULTS: Totally 628 NIH and ICR mice were involved in result analysis. ① The level of IgG and CIC of mice in the model group was significantly higher than that in the other 3 groups respectively (P ＜ 0.01),while the level of C3 was significantly lower than that in the other 3 groups (P ＜ 0.05 to 0.01). ② The swelling degree of mice in the diclofenac group and Xiaohuoluo pills group was significantly lower than that in the blank control group respectively ( both P ＜ 0.01). ③ The rosette rate of RBC-C3b receptor and RBC immune compound in the blank control group was significantly higher than that in the other 2 gro ups respectively (P ＜ 0.01). ④ The phagocytic index (K value )in the diclofenac group and Xiaohuoluo pills group was significantly lower than that in the blank control group, respectively (both P ＜ 0.01).⑤ IgM-type HC50 and the level of serum MDA of CY group and Xiaohuoluo pills group were obviously lower than those in the immune control group (P ＜ 0.01),while the level of C3 was higher than that of immune control group, there was no significant difference in the activity of serum SOD between CY group or Xiaohuoluo pills group and immune control group (P ＞ 0.05). ⑥The ratio of agar granulation tissue mass to body mass in the diclofenac group or Xiaohuoluo pills group was significantly lower than that in the model group(P ＜ 0.01).⑦ The times of distortion of mice within 20 minutes in the diclofenac group or Xiaohuoluo pills group were signifi cantly less than those of model group(P ＜ 0.01,0.05).⑧The ear swelling degree of dimethylbenzene-induced inflammatory models and croton oil-induced inflammatory models,and foot swelling degree of carrageenan-induced acute inflammatory models as well as the level of prostaglandin E in the inflammatory exudates in the diclofenac group were significantly milder or lower than those in the model group(P ＜ 0.05 to 0.01),and the level of prostaglandin E in the inflammatory exudates in the Xiaohuoluo pills group was significantly lower than that in the model group (P ＜ 0.01).CONCLUSION: Xiaohuoluo pills possess pharmacological effects of immunosuppression, anti-proliferative inflammation, analgesia and antioxidation.