Research On The Immunoregulation Of Sodium Houttuyfonate Homologues On Macrophage

Houttuynia Cordata is an important medical plant of the Saururaceae family with antibacterial activity and dephlogistication.Houttuynin(Decanoyl acetaldehyde)is the effective component with antibacterial activity.Sodium houttuyfonate produced through chemical synthesis,modifying the aldehyde group with NaHSO₃,is used as clinical medicine extensively for the houttuynin's low content in Houttuynia Cordata, indissovlabe in water and instability of chemical property.It is indicated by modern pharmacological research that houttuynin has the functions of not only antibacterial activity and dephlogistication but also immunoregulation.It is shown in many animal test in vivo and clinical application that sodium houttuyfonate likely promote the specific and nonspecific immunity.The experiment results in vitro demonstrate that sodium houttuyfonate could raise the IL-2 level in T lymphocyte media and activate the macrophage,promoting phagocytosis,the production of the acid phosphatase,lysozyme and so on,the hightening of respiratory burst and the rising of intracellular free calcium ion.The purpose of this research is to investigate the immunoregulation on macrophages of sodium houttuyfonate homologues(HOU-C₈:nonanoyl acetal sodium sulfite; HOU-C₁₀:decanoyl acetal sodium sulfite;HOU-C₁₂:lauroyl acetal sodium sulfite; HOU-C₁₄:tetradecanoyl acetal sodium sulfite;HOU-C16:hexadecanoyl acetal sodium sulfite)and the relation of the immunoregulation and the concertration or the length of carbon chain.It is detected firstly the activities of lysozyme secreted by macrophages co-cultured with the sodium houttuyfonate homologues(HOU-Cn,n=8,10,12,14,16).The lysozyme in serum is mainly secreted by macrophages.The lysozyme specific activities are determined after culturing macrophages with different concentrations of sodium houttuyfonate homologues(the final concentrations are as bellow:0μg/mL,0.50μg/mL, 1.0μg/mL,2.0μg/mL,4.0μg/mL,8.0μg/mL,16μg/mL,32μg/mL,64μg/mL)for 24 hours. Then,we detected the lysozyme activities after the lysozyme molecule binding with HOU-Cn to make sure whether or not sodium houttuyfonate could influence the lysozyme activity.The fluorescence spectra emited by the Tyr residue and the Trp residue are scaned to describe whether or not the lysozyme comformation changes when HOU-Cn has bound to lysozyme molecule.Acid phosphatase(ACP)works in the lysosome of macrophage,and degrades the antigen phagocytosized.ACP in the lysosome of macrophage is tartrate-resistant acid phosphatase(TRAP).Macrophages are cultured with different concentrations of HOU-Cn(n=8,10,12,14,16)for 24 hours,the final concentrations of HOU-Cn being 0μg/mL,0.50μg/mL,1.0μg/mL,2.0μg/mL,4.0μg/mL,8.0μg/mL,16μg/mL,32μg/mL, 64μg/mL.Wash the macrophges with fresh 10%NBS RPMI-1640 media 3 times,add the fresh 10%NBS RPMI-1640 media,and split the macrophages by freeze thawing 3 times,then detect the TRAP specific activities in the supernatant.The reactive oxygen intermediates(ROI)are produced in the macrophage repiratory burst.ROI could work intracelluarly,and be secreted extracelluarly,which is involved with degrading the antigen and killing the tumor cell.The fluorescence intensity at 525nm is measured with DCFH-DA after treated with diffenent concerttations HOU-Cn, which displays indirectly the macrophage respiratory burst.Calcium ion functions in some intracellular signal pathways,and plays a key role in cell's physiological functions regulation.The intracellular calcium concentrations are determined with Fura-2 after macrophages mixed with different concentrations of HOU-Cn solution containing or not containing 1.5mmol/L neomysine.The change of intracellular calcium concentrations could show the effect of the concentration of HOU-Cn on the intracellular calcium concentration in macrophages,and whether or not the neomysine inhibiting phospholipase C(PLC)could affect the release of intracellular calcium ion.In the range of 0～2.0μg/mL,the lysozyme specific activity in macrophage media could rise generally with the increasing of HOU-Cn(n=8,10,12,14,16) concentration,or with the extending of carbon chain of HOU-Cn(n=8,10,12,14,16). HOU-Cn(n=8,10,12,14,16)could also inhibite the lysozyme activity.The lysozyme comformation changes when HOU-Cn(n=8,10,12,14,16)binds to lysozyme by hydrophobic interaction,so that the lysozyme activity decreases.In the range of 0～2.0μg/mL,the TRAP specific activity in macrophage media could rise with the increasing of HOU-Cn(n=8,10,12,14,16)concentration.In the range of 16～64μg/mL,the TRAP specific activity in macrophage media appares an ascending tendency with the extending of carbon chain of HOU-Cn(n=8,10,12,14,16).HOU-Cn(n=8,10,12,14,16)could heighten the macrophage respiratory burst without any antigen(such as ConA),and promote the production of ROI.HOU-Cn(n=8,10,12,16) could promote significantly the macrophage respiratory burst in the range of 0～16μg/mL which is dose dependent.The respiratory burst of macrophages treated with 0.50～64μg/mL HOU-C₁₄are all significant higher than that of macrophages as control, and there is no significant difference in respiratory burst in the range of 0.50～64μg/mL HOU-C₁₄.By activating PLC,HOU-Cn(n=8,10,12,16)promote the production of IP₃,which turns on the calcium ion channel on the endoplasmic reticulum(ER)and increases the intracellular free calcium ion concentration in macrophages.It seems to be the unique way to release the intracellular calcium ion from ER into cytoplasm.The calcium ion is necessary for respiratory burst in macrophage,which arise from the ascending of the intracellular free calcium ion concentration.As the key signal in a few intracellular signal pathways,some genes expression relative to immunological function could arise from the ascending of the intracellular free calcium ion concentration.In the range of 0～16μg/mL of HOU-Cn(n=8,10,12,14),the effect of releasing the intracellular calcium ion becomes stronger and stronger with the increasing of HOU-Cn(n=8,10,12,14) concentration,or with the extending of carbon chain of HOU-Cn(n=8,10,12,14). HOU-C₁₆displays a descending tendency on the effect of releasing the intracellular calcium ion,and no significant influence on this effect in the range of 16～64μg/mL.The primary experimental research demonstrates,HOU-Cn(n=8,10,12,14,16)should raise the activity of lysozymc and TRAP produced by macrophages,which could promote the degradation of foreign matter in phagocytotic vesicle and out side of cell, and HOU-Cn(n=8,10,12,14,16)should up-regulate the intracellular signal pathway involved with calcium ion to promote some genes expression relative to immunological function,and heighten the respiratory burst by raising the concentration of intracellular calcium ion to promote the production of ROI which benefits the immunological function improvement of macrophages.There is some regulation between the immunoregulation and the carbon chain length of HOU-Cn(n=8,10,12,14,16).