| IntroductionScoparone ( Sco) is one of the major ingredients of Chinese crude drug " herba artemisiae capilloais" , whose pharmacological actions include many aspects. It has widely values, abundant resources, and fine prospect of development.It has been found in our past study that Sco can directly relax trachea! smooth muscle, and has anti - asthmatic effect on asthmatic models of guinea pigs. Airway infiltration by inflammatory cells, particularly eosinophils and activated T lymphocytes, is one of the characteristic features of asthma. Interleukin - 2 (IL - 2) plays a key role in lymphocyte proliferation. In this experiment, we studied the effect of Sco on lymphocyte proliferation and IL - 2 production of asthmatic guinea pigs in vitro using the MTT assay.Methods1. The guinea pigs were divided into asthmatic group and control group at random, the asthmatic group were sensitized and induced asthma with ovalbu-min.2. Measurement of lymphocytes proliferation;The spleen were removed aseptically, placed in Hanks (PH7. 2) , teased with a scapel and strained though a mesh grid, and the resting cells were centri-fuged. For splenocytes, lysis of red blood cells were accomplished by resuspend-ing the cells in Tris ammonium chloride. The viability was determined by trypan blue exclusion. Splenocytes were suspended at 2 106/ml in RPMI - 1640 me-dium. In 96 - wells tissue culture plates, 100 l splenocytes were incubated with PHA, various concentrations of Sco or Dex. After incubations for 48 hours, 10 l MTT was added to each well, after 4 hours later, 150 l DMSO was added and cell proliferation was measured using the MTT assay. 3. IL-2 assay:(1) Induction of IL - 2: After a 48 - h incubation with Sco , Dex and Co-nA or medium alone, lymphocytes were removed by centrifugation, and the culture medium was collected and stored frozen ( -20 C ).(2) Preparation of effect cells: Spleen single - cell suspensions were prepared and ConA was added. Cells were collected after 48 hours and washed twice with Hanks. Then cells were suspended in complete culture medium.(3 ) Measurement of IL - 2: 100 l supernatants with IL-2 and 100 l effect cells were added to 96 - wells tissue culture plates and IL-2 activity was assayed on IL-2 dependent effect cells. Human recombinant IL-2 served as the standard.The data are expressed as means S. E. M. Statistical significant differences were determined with student's t - test. Values of P <0. 05 was considered significant.Results1. Effect of Sco on proliferation of spleen lymphocyte of guinea pigs: Sco could suppress the proliferation of spleen lymphocyte of asthmatic guinea pigs from 10 -5M/L to 5 10 -4M/L in the matter of dose dependent. Dex (10-6 mol L-1 and 5 10-6mol L-1) also has suppressive action on lymphocyte proliferation(P <0.01, compared with normal control). The suppressive rate of Sco on asthmatic groups is higher than normal groups (IC503. 98 10-4vs. 5. 01 10-4 mol L-1 ,P <0. 05). Sco also suppress the T lymphocyte cell proliferation induced by PHA from 5 10 -5 ~ 3 10 -4mol L-1 ( P < 0. 01, compared with normal control).2. Effect of Sco on IL-2 production: The IL-2 activity in asthmatic guinea pigs were significantly increased compared with normal control group ( p<0.05 ). Sco(5 10 -5mol L-1 ~ 5 10 -4mol L-1) could dose dependently reduce the IL -2 activity in asthmatic guiea pigs. Dex (5 10-6 mol L-1) reduce IL -2 production in asthmatic ones also (p <0.05).DiscussionDisordered immune reaction in patients with asthma is mainly characterized by lymphocyte proliferation. The role played by T lymphocytes in the pathogene-sis of asthma is more important. Sco is one of the major ingredients of " herba ar-temisiae capilloais" , in this study, we established the OVA - induced asthmatic model of guinea pigs, the results indicated that Sco could suppress the lymphocytes liferation of asthmatic guinea pigs directly, and suppress the T lymphocytes proliferation induced by PHA in asthmatic guinea pigs. Sco had m... |
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