Treating And Mechanical Study Of Bupleurum Polysaccharides On Acute Lung Injury

The pathological characteristics of acute lung injury(ALI,a pulmonary inflammation and complication of increased lung permeability) are diffused alveolar-capillary membrane injury and pulmonary edema,which are caused by severe infection,trauma,shock,et al.ALI is the early phase of ARDS(acute respiratory distress syndrome).Its main pathophysiological changes include the reduced lung compliance,the increased intrapulmonary shunt fraction as well as the imbalance of ventilation/perfusion ratio.And the clinical manifestations are refractory hypoxemia,accelerated respiratory frequency and respiratory distress.Lung is the primary target organ in the systematic inflammatory response caused by serious injuries,which may lead to MODS(multiple organ dysfunction syndrome) as the severe stage of the pathological process.ALI is the most common change in the development of MODS.Bupleurum smithii(B.smithii,Umbeliferae),a perennial herbaceous plant,is one of the most frequently prescribed crude herbs in the prescriptions of traditional Chinese medicines.The dried root is used as Radix Bupleuri.Also known as "Chai-Hu",B.smithii is also used for the treatment of inflammatory diseases,and auto-immuhe diseases.Some saikosaponins from B.smithii were reported as the major bioactive components with anti-inflammatory activity and the activity of re-modulating immune system.However,there are relatively few reports of B.smithii for treating ALI.Recent researches suggest that inappropriate activation of complement is involved in the pathogenesis of ALI;meanwhile,B.smithii has been reported to possess the anti-complementary activity.The present study aims to explore the therapeutic effects of the Bupleunim polysaccharides in treating ALI,as well as its influence on SOD activity and MDA content both in serum and BALF (bronchoalveolar lavage fluid).In addition,further experiments indicated the underlying mechanism by investigating the possible effect of Bupleurum polysaccharides on the function of leukocytes. Part 1:Complement inhibition and Anti-oxidative effect of the Bupleurum polysaccharides in treating acute lung injuryAIM To explore the complement inhibition and anti-oxidative effect of Bupleurum polysaccharides,when it was used in treating acute lung injury of rats.METHODS Thirty—five healthy male Sprague-Dawley rats were randomly divided into seven groups:sham group,model control group,vehicle group,small dose group,median dose group,large dose group and prednisolone acetate group.The "two-hit" lung injury model characterized by hemorrhagic shock with resuscitation,followed by an intratracheal LPS administration,was established.The activity of serum complement was tested.The malondialdehyde(MDA) and the activity of superoxide dismutase (SOD) in the serum and BALF were assayed.The severity of lung tissue injury was evaluated under light microscope.RESULTS Along with time,the contents of MDA in the serum of every group was increased,and the activity of SOD and activity of complement were decreased.Compared with the model groups,the contents of MDA in the serum and the BALF of median and large dose groups were significantly decreased at 4h(P＜0.05 and P＜0.01,respectively),and the activity of SOD was significantly increased at 4h(P＜0.05 and P＜0.01,respectively).However, among the median and large dose group and prednisolone acetate group,there was no significant difference(P＞0.05).Under optical microscopes,the lung tissue of sham group was normal.The injury of lung tissue of the model group,the vehicle group and small dose group was obvious.But the injury of lung tissue of the median,large dose group and prednisolone acetate group was slight.The activity of serum complement among groups was no significant difference(P＞0.05) at Oh and 2h.The activity of serum complement of the model groups and small dose groups were significantly decreased at 4h.However,the median and large dose group can inhibit the decreasing of serum complement.CONCLUSION The Bupleurum polysaccharides can treat acute lung injury,probably by influencing the anti-oxidation and complement inhibiton.Part 2:Effect of the Bupleurum polysaccharides on the phagocytic function of macrophageAIM To study the effect of the Bupleurum polysaccharides on the chemotaxis of leukocytes.METHODS Mouse peritoneal leukocytes were separated and randomly divided into 3 serieses(12 groups):1.The control series,including the SRBC group (sheep red blood cell group),the blank control group(RPMI-1640 group),the chemokine group(positive control,IL-8,50ng/ml) and the group of complement activated products(10%,v/v);2.The drug series without the complement activated products,including the low dosage group(0.8mg/ml),the medium dosage group (1.6mg/ml) and the high dosage group of the Bupleurum polysaccharides,as well as the prednisolone acetate group(4mg/ml);3.The drug series with the complement activated products,including the low dosage group(0.8mg/ml),the medium dosage group(1.6mg/ml) and the high dosage group of the Bupleunun polysaccharides,as well as the prednisolone acetate group(4mg/ml).Every group contained 5 samples (n=5).The macrophages of every group were cultured for 1h at 37℃in the 5%CO₂ incubator.The supernatant was obtained for the assay of MDA content and SOD activity.The phagocytosis of macrophages was observed with the microscope.The phagocytic percentage was calculated.RESULTS There were no significant differences in SOD activity,MDA content and phagocytic rate among the drug groups without complement activated products(P＞0.05).Compared with the blank control group,the SOD activity of the group of complement activated products were significantly decreased and its MDA content increased(P＜0.01),meanwhile the phagocytic function of macrophages enhanced obviously(P＜0.05).The data showed no differences compared with the low dosage group(P＞0.05).SOD activity and the phagocytic rate of the high and the medium dosage groups were significantly higher, MDA content lower than those of the complement activated products group.CONCLUSION The Bupleurum polysaccharides may act as an anti-oxidant and may facilitate the phagocytic function of macrophages enhanced by complement activated products.Part 3:Effect of the Bupleurum polysaccharides on the chemotaxis of leukocytesAIM To study the effect of the Bupleurum polysaccharides on the chemotaxis of leukocytes.METHODS Mouse peritoneal leukocytes were separated and randomly divided into 3 series(12 groups):1.The control groups,including the SRBC group (sheep red blood cell group),the blank control group(RPMI-1640 group),the chemokine group(positive control,IL-8,50ng/ml) and the group of complement activated products(10%,v/v);2.The drug groups without the complement activated products,including the low dosage group(0.8mg/ml),the medium dosage group (1.6mg/ml) and the high dosage group of the Bupleurum polysaccharides,as well as the prednisolone acetate group(4mg/ml);3.The drug groups with the complement activated products,including the low dosage group(0.8mg/ml),the medium dosage group(1.6mg/ml) and the high dosage group of the Bupleurum polysaccharides,as well as the prednisolone acetate group(4mg/ml).Every group contained 5 samples (n=5).The macrophages of every group were cultured for 1h at 37℃in the 5%CO₂ incubator.0.6ml of the supernatant was obtained and added into the lower Transwell chamber(the polycarbonate membrane with the pore diameter of 0,8μm).0.1ml of the leukocyte suspension(1.0×10⁶ cells/ml) was added into the upper chamber.Then, Transwell chamber was incubated for 3h at 37℃in the 5%CO₂ incubator.The chemotaxis of leukocytes was observed with the microscope and characterized by the calculated migration percentage.RESULTS Compared with 1640 group,the migration percentage of the SRBC group showed no difference,while that of the IL-8 group was significangly higher(P＜0.01).There were no significant differences between the drug groups without the complement activated products and the blank control group(P＞0.05).The data suggested that complement activated products would obviously increase the migration rate of leukocytes,similarly with the effect of IL-8.The Bupleurum polysaccharides may inhibit the migration of leukocytes caused by complement activated products in a dose-dependent manner.And there were significant differences in the migration percentages of the high,the medium dosage groups and the control group(P＜0.01).CONCLUSION The Bupleurum polysaccharides may inhibit the chemotaxis of leukocytes.