| Objective:To investigate the effects of Tong-Bi-Ling (TBL) and its different pharmacologically active extracts on the T cells activation by polyclonal activators with methods of in vitro and serum immunopharmacology for the application of this drug clinically. In the same time, to establish a system for assessment of multi-components drugs with activity of immunity regulation, which would provide a tool for the investigation on multi-components drugs with action of immunity regulation, including TBL. Methods:(1) After separation of the lymphocytes from lymphoid nodes of C57BL/6 mouse, some cells were preincubated with CHX, CsA, n-Butanol soluble extract, ethyl acetate soluble extract, chloroform soluble extract, total alkaloids, water soluble extract of Tong-Bi-Ling respectively for a hour, then further incubated with polyclonal activators (Con A or PDB); The other cells were preincubated with total alkaloids of TBL in different concentration respectively for a hour, then further incubated with polyclonal activators (Con A or PDB); (2 ) After separation of the lymphocytes from lymphoid nodes of C57BL/6 mouse, these cells were preincubated with serum containing CHX, serum containing CsA and serum containing TBL respectively for a hour, then further incubated with polyclonal activators (Con A or PDB).Harvesting the cells after whole incubation for 24h, we estimated the expression rates of CD69 on T cells by flow cytometry following two-color immunofluorescent staining.Results:(1) The expression rates of CD69 on the T cells preincubated with chloroform soluble extract, total alkaloids of TBL stimulated by Con A were (55.38± 2.24)%, (44.47± 2.90) %, which showed significant difference with the expression rate of the control groups [ (65.47± 1.67)%] (P<0.01); and the expression rate of CD69 on the T cells preincubated with chloroform soluble extract, total alkaloids of TBL stimulated by PDB were(77.66± 1.01)%, (69.11± 2.14)%, which didn't show significant difference with the expression rate of the control groups [ (86.63± 3.77) %] (P>0.05). It showed potent inhibition of chloroform soluble extract, total alkaloids of TBL on the CD69 expression on activated T cells in vitro.(2) The expression rate of CD69 on the T cells preincubated with total alkaloids of TBL stimulated by Con A were(68.70± 2.59)%, (65.72± 1.81)%, (59.39± 2.01)%, (54.95± 1.75)%, (29.54± 3.22)% respectively, according to the concentration from 0mg/L, 50mg/L, 100mg/L, 200mg/L to 400mg/L. The effects of total alkaloids to down-regulate CD69 expression on T cells activated by Con A gradually strengthened. Likewise, The expression rate of CD69 on the T cells preincubated with total alkaloids stimulated by PDB were (91.00± 1.60)%, (86.91± 0.58)%, (76.15± 6.18)%, (71.72± 1.42)%, (27.67± 2.54)% respectively. The inhibition effects of total alkaloids on CD69 expression in responsed to PDB also gradually strengthened.(3) The expression rate of CD69 on the T cells preincubated with seurm containing TBL after the stimulation in response to Con A was (54.67± 6.69)%, which showed significant difference with the expression rate of the control groups[(64.35±10.13)%] (P<0.01); Whereas the expression rate of CD69 on the T cells preincubated with serum containing TBL after the stimulation in response to PDB was (80.99± 6.18)%, which didn't showsignificant difference with the expression rate of the control groups[(79.68±4.17)%](P>0.05). Conclusion:The total alkaloids and chloroform soluble extract of TBL potently inhibited activation of mouse T cells in vitro, and the inhibition effects of total alkaloids was in a dose-dependant way. In clinical dose, 5% serum containing TBL showed inhibitory effects on the activation of T cells in response to stimulation of Con A except that of PDB. Our data suggested that the inhibitory effects on the abnormal activation of T cells during the pathgenesis and progression of rheumatoid arthritis may be one of the important mechanisms applied to the treatment of the disease. W...
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