A With The Pla <sub> 2 </ Sub> Activity Inhibition Of Herbal Medicine Screening And Mechanism Study

Phospholipase A2(PLA2,EC.3.1.4), which was a sort of important enzyme in metabolism and regulation, represented a growing family of enzymes hydrolyzed glycerophospholipids at the sn? position to produce free fatty acids including arachidonisc acid(AA), a precursor of bioactive eiosanoids, and lysophospholipids. PLA2 had been implicated in various cellular responses, such as signal transduction, host defense, blood coagulation, digestion and membrance remodeling. The founction of PLA2 in inflammatory process was more focused on. Because PLA2 was found in almost every tissue and involved in normal physiologic function, it was important and difficult to find a inhibitor of PLA2, which inhibited the harmful action of PLA2 in inflammation and did not influenced the physiological role of PLA2. Considering the nature and few side effect property of traditional chinese medicine, we put interest to screen out some traditional chinese medicines with above inhibitory effect on PLA2 activity. This study included three parts as following: l.To establish a method, which was reliable, stable, simple and could detect both sPLA2 (secretory phospholipase A) and cPLA2 (cytosolic phospholipase A) activity. E. coli membranes doped by the [3H1 labeling arachidonate was prepared and hydrolyzed by PLA2 in certain condition, and the enzyme activity was expressed with the hydrolyzing rate. Intra-day coefficient of variation (CV) of cPLA2 was 5. 2% and inter-day CV was 10.9%, and 4.9% and 7.8% for sPLA2, respectivily. Results of multiple proportion dilution assay showed a good linear relationship. Serum sPLA2 activities of patients with acute cholecystitis were significantly higher than that of nomal control group. There was a significantly difference of activities of sPLA2 and cPLA2 between the LPS induced leukemia cell K562 and control. This method is specific, stable and sensitive, it may be used in clinic and scientific research. 2. To screen the traditional chinese medcines which were the inhibitor of PLA2 In this investigation, the acute inflammation model rat was produced by LPS i. p. After treatment with different doses of Berberine, Gynostemmn pentaphyllum Makino saponin, Matrine and extract of Ginkgo bilobaL leaves, the serum sPLA2 activities in the acute inflammation model rats were tested at different time with 1?4h. The 0. 5mg/kg, 1mg/kg and 2mg/kg extract of Ginkgo biloba L leaves could significantly inhibit the activity of serum sPLA2 at examined time, and 0. 5mg/kg group showed the highest inhibitory rate (72.8?.6%,n5)at lh,but the drug toxic effects was appeared at the dose of 3mg/kg. The 15mg/kg, 30mg/kg, 60mg/kg and 120mg/kg Matrine could significantly inhibit the activity of serum sPLA2 at examined time, and 30mg/kg group showed the highest inhibitory rate Berberine had a little inhibition of PLA2, At one or four hours after 75mg/kg intragastrically. But treatment of Gypennoside had abvious drug toxic effects at once, so we abandoned it. The above results suggested Ginkgo biloba L, Matrine and Berberine had effects of sPLA2 inhibition. 3. To study the anti-inflammation action and PLA2 activity inhibition mechanism of Matrine. The activities of sPLA2 in serum and cPLA2 in leucocyte were measured with the established chemoradiation method, and the cytosolic free Ca²⁺ le...