Effects Of Bupleurum Chinensis Polysaccharides On Secretion And Chemotaxis Of Macrophages

Radix Bupleuri (umbelliferae perennial herb), known as ’Chai-Hu’, is one of the most frequently prescribed crude herbs in the prescriptions of traditional Chinese medicine. The modern pharmacology studies have shown that Bupleurum has anti-inflammatory, analgesia,cooling and other functions. Previous studies in our lab demonstrated that the crude polysaccharide isolated from Bupleurum chinense (BPs) had a beneficial effect on acute lung injury (ALI). But, the specific mechanism is still unclear.Macrophages play important roles in both innate and adaptive immune systems, and involve the pathogenesis of varied disease including autoimmune disease and systemic inflammatory response. For years, RAW264.7 cells and mouse peritoneal macrophages have been used to dissect the complex functions of macrophages. However, MPMs and RAW264.7 cells have distinct characteristics. The differences between these cells have not been quite clear and are often ignored by researchers. Furthermore, LPS contamination is inevitable in natural extracts and fails to gain enough attention.In this study, we examed cytokines secretion and TLR4 protein expression for the purpose of determining the heterogeneity between RAW264.7 cells and MPMs as regards responding to LPS. The abilibity of polymyxin B to abolish LPS biological properties was tested in RAW264.7 cells and MPMs in order to use it to eliminate the interference caused by LPS contamination. Effects of Bupleurum chinense polysaccharides were studied on MPMs and RAW264.7 cells.1. Comparison of responses of mouse peritoneal macrophages and RAW264.7 cellsAim:To determine some differences in response to LPS stimulation between RAW264.7 cells and MPMs, so as to provide the basic information for investigators to decide which macrophage type is appropriate, especially in studies involving LPS.Methods:(1)LPS(0.01,0.1,1,10,100 and 1000 ng/mL)were added to stimulate MPMs and RAW264.7 cells for 24 h, NO and TNF-a productions were tested.(2) LPS(10,100 and 1000 ng/mL) were added to stimulate MPMs and RAW264.7 cells, TLR4 protein expressions were tested. (3) Polymyxin B (0.01,0.1,1,10,30 and 100 μg/mL) were added to MPMs and RAW264.7 cells for 24 h,the toxicity of PmB was measured. (4) RAW264.7 cells were stimulated with 10 ng/mL LPS for 10 min and 20min, then PmB was added; PmB was added into the RAW264.7 cells for 10 min and 20min, then 10 ng/mL LPS was added to stimulate the RAW264.7 cells; LPS was incubated with PmB for 10min,20min, 1h,2h,5h and 24h, then the mixture was used to stimulate RAW264.7 cells. LPS was incubated with PmB for 1h,5h and 24h, and then the mixture was used to stimulate MPMs. The final concentrations of PmB were 0.01,0.1,1,10,30 and 100μg/mL. As for MPMs, LPS final concentration was 1000 ng/mL. As for RAW264.7 cells, LPS final concentration was 10 ng/mL. The cells were cultured for 24h and the ability of PmB to block LPS biological activities was tested. (5) Bupleurum chinensis polysaccharides (BPs) were pre-incubated with PmB for 24 h, and then added to MPMs and RAW264.7 cells for 24h. The final concentrations of BPs were 10,40 and 160 μg/mL while PmB final concentration was 30μg/mL. The action of same concentration of normal BPs was also tested.Nitric oxide (NO) were quantified by Griess reation, cytokines productions were measured by ELISA, TLR4 protein expression was determined by Western blot and immunofluorescence microscopy.Results:(1) NO and TNF-a production significantly increased in RAW264.7 cells when LPS≥0.1 ng/mL (p<0.05) and in MPMs when LPS≥00 ng/mL(p<0.05) The amounts of NO and TNF-a in a given LPS concentration were greater in RAW264.7 cells than in MPMs (p<0.05). (2) The expression of TLR4 protein was detected in naive RAW264.7 cells, but it was almost undetectable in MPMs. The expression levels were increased in RAW264.7 cells stimulated by LPS in a concentration-dependent manner. In MPMs, an increase in TLR4 expression was only observed when treated with 1000 ng/mL LPS. (3)Only 100μg/mL PmB was cytotoxic to MPMs and RAW264.7 cells. (4)After the cells were stimulated by LPS for 10 and 20 min, PmB at all given concentrations could not effectively inhibit NO release. When PmB was added to cells 10 or 20 min before LPS stimulation, NO production was significantly reduced (p<0.05). The maximal inhibition rate was 39%. When LPS was pre-treated with PmB, NO and TNF-a secretion from RAW264.7 cells and MPMs were significantly reduced (p<0.001). The inhibitory effects were time-and dose-dependent. (5) BPs significantly augmented NO production of RAW264.7 cells and MPMs at all given concentrations. When the drug was pre-incubated with 30μg/mL PmB for 24 h, NO production was decreased.Conclusion:RAW264.7 cells responded more sensitively to LPS than MPMs did. Such differences must be taken into consideration, especially in studies involving LPS. Besides, in order to reliably identify immunomodulatory molecules using RAW264.7 or MPMs, LPS contamination must be cleaned by polymyxin B or other methods appropriately.2. Effects of Bupleurum chinensis polysaccharides on mouse peritoneal macrophages and RAW264.7 cellsAim:To investigate effects of BPs on secretion and chemotaxis of MPMs and RAW264.7 cells.2.1. Effects of BPs on the secretion of proinflammatory cytokines by MPMs and RAW264.7 cells.Method:MPMs and RAW264.7 cells were assayed as follows. (1) BPs were pre-incubated with PmB for 24 h, and then added to MPMs and RAW264.7 cells for 24 h. The final concentrations of BPs were 10,40 and 160 μg/mL while PmB final concentration was 30μg/mL. Cytokines productions were measured. (2) BPs were pre-incubated with PmB for 24 h, and then added to MPMs and RAW264.7 cells together with LPS for 24h. The final concentrations of BPs were 10,40 and 160 μg/mL while PmB final concentration was 30 μg/mL. As for MPMs, LPS final concentration was 1000 ng/mL. As for RAW264.7 cells, LPS final concentration was 10 ng/mL. Cytokines productions of LPS-stimulated cells were measured. Nitric oxide(NO) were quantified by Griess reagent, cytokines productions were measured by ELIS A.Results:(1)As for MPMs and RAW264.7 cells, BPs enhanced the secretion of NO, TNF-α,IL-1βå'Œ IL-6 production. (2) As for LPS-stimulated MPMs,40,160 μg/mL BPs dramatically suppressed NO, TNF-α, IL-6 and IL-1β production (p<0.05). As for LPS-stimualted RAW264.7 cells,160 μg/mL BPs futher promoted secretion of NO, TNF-a and IL-6 (p<0.05)Conclusion:BPs promoted the secretion function of MPMs, but suppressed cytokines production of LPS-stimulated MPMs; BPs promoted the secretion function of RAW264.7 cells and LPS-stimulated RAW264.7 cells.2.2. Effects of BPs on the chemotaxis of MPMs and RAW264.7 cellsMethods:MPMs and RAW264.7 cells were assayed as follows. (1) BPs were pre-incubated with PmB for 24 h, and then added into lower chamber of transwell. MPMs and RAW264.7 cells were added into upper chamber and incubated for 4 h. The final concentrations of BPs were 10,40 and 160 μg/mL while PmB final concentration was 30 μg/mL. (2) BPs were pre-incubated with PmB for 24 h, and then added to lower chamber together with C5a. MPMs and RAW264.7 cells were added into upper chamber and incubated for 4 h. The final concentrations of BPs were 10,40 and 160 μg/mL. PmB final concentration was 30 μg/mL. C5a final concentration was 50 ng/mL. Effects of BPs on the chemotaxis of MPMs and RAW264.7 cells were measured. Results:(1) BPs enhanced the chemotaxis of MPMs and RAW264.7 cells.160 μg/mL had a significantly difference (p<0.05). (2)BPs inhibited the chemotaxis of MPMs stimulated by C5a, but enhanced the chemotaxis of RAW264.7 cells stimulated by C5a.Conclusion:BPs could enhance the chemotaxis of MPMs and RAW264.7 cells. When cells were stimulated by C5a, it could further promote the chemotaxis of RAW264.7 but suppress that of MPMs.