The Study Of The Immunomodulatory Activity From Sulfur-fumigated And Non-fumigated Smilax Glabra Roxb.

ObjectiveSulfur-fumigation as a kind of storage method to anti-mildew,anti-corrosion and insect-resistant has both advantages and disadvantages.This research was made sulfur-fumigated Smilax glabra Roxb.and extract,separated and purified the sulfur-fumigated Smilax glabra Roxb.crude polysaccharides(SSGRP)and non-fumigated Smilax glabra Roxb.crude polysaccharides(NSGRP).The difference of physicochemical property between SSGRP and NSGRP were characterized and compared.The study mainly revealed the sulfur-fumigation toxicity on immune cells and it could reduce the immunomodulatory activity of the herbs,and explored its mechanism.To fill the gaps of sulfur-fumigation on the immune research,provided a scientific basis and reference to abolish the sulfur-fumigation method.Methods1.Made sulfur-fumigated S.glabra and refluxed with boiled water extract,through ethanol precipitation and Sevage method to separate and purify the SSGRP and NSGRP.2.Compared with SSGRP and NSGRP from the external appearance,dissolvability(water,trichloroacetic acid,methanol,ethanol,ether and acetone)and extraction yield of crude polysaccharide.The glucose levels of the polysaccharide samples were evaluated by the common phenol-sulfuric acid method,determined the inorganic elements by ICP-AES and the FT-IR spectrum scan.3.The effects of SSGRP and NSGRP on the macrophages cell viability were measured by MTT method.4.The effect of SSGRP and NSGRP on the immunomodulatory activity of macrophages were studied by below methods.The secretion of TNF-? and IL-6 cytokines in the macrophages cell supernatant were detected by ELISA kit;the gene expression of TNF-? and IL-6 were measured by RT-PCR.5.The effects of SSGRP and NSGRP on the Mouse spleen lymphocytes viability were measured by CFSE proliferation experiment.6.The effects of SSGRP and NSGRP on the different types of Mouse spleen lymphocytes were measured by flow cytometry analysis.7.To determine whether the SSGRP promote macrophages apoptosis by the methods as follows:Hoechst 33258 staining,Annexin V and PI double-stained cells,cell cycle and Mitochondrial membrane potential(MMP).8.The effect and mechanisms of SSGRP and NSGRP on promote macrophages apoptosis and reduce the immunomodulatory activity of macrophages were studied by below methods.The apoptosis related factor gene expression of Bax and Caspase-8 were measured by RT-PCR;the expression of Bax and Caspase-8 protein and MAPK signal pathways were detected by Western blotting.Results1.Sulfur-fumigated S.glabra through the steps of "Water extract?Ethanol precipitation-Sevage method-Freeze drying" obtained the SSGRP 20.85 g and the NSGRP 6.35 g,the extraction yield was 4.17%and 1.27%,respectively.2.The external appearance of S.glabra was bleached brighten and whiten through sulfur-fumigation.The dissolvability between the SSGRP and NSGRP in different dissolvents made a great difference.3.The UV spectrum scan of both the SSGRP and NSGRP were measured with maximum absorption peak near 490 nm,so 490 nm is adopted as the maximum absorption wavelength.The results of the phenol-sulfuric acid method showed that the glucose of SSGRP and NSGRP were 46.43%and 42.18%,respectively.The SSGRP increased 4.25%.4.The SSGRP sulfur element was two times higher than NSGRP,the carbon and hydrogen element were the same,the results showed that sulfur-fumigation would made the sulfur dioxide residue increased.5.SSGRP and NSGRP were similar in the FT-IR spectrum scan.But in the wavelength range of 3400-2900 cm-1,there was a little difference,meaned the polysaccharide structure might be change.The absorption peak at 1700-1300 cm-1 was sharp,at 1652.7 cm-1 was shifted to the left of the SSGRP.And at 1160-900 cm-1,the absorption peak was sharp and the intensity was weaker than NSGRP,meaned the sulfur-oxygen double bond might be change after sulfur-fumigation.6.We used the MTT assay to investigate the anti-proliferative potential of SSGRP and NSGRP on RAW 264.7 cells,the results showed that the cell viability of SSGRP decreased from 93.18± 1.67%to 60.95 ± 1.77%in a dose-dependent manner within the concentration of 1000-8000?g/mL.But NSGRP has no significant effect on the growth of macrophages.The results revealed that sulfur-fumigation made the non-toxic S.glabra crude polysaccharides to produce certain cytotoxicity.7.In the ELISA assay,NSGRP can remarkably increase the secretion of TNF-? and IL-6 of macrophages with the great immunomodulatory activity.The RT-PCR assay was the same as the increment of TNF-? and IL-6 gene expression of NSGRP.The expression of the SSGRP group was obviously lower than NSGRP,although SSGRP can increase the secretion of TNF-? and IL-6 too.The results showed the mechanism of sulfur-fumigation was decreased the cytokines gene expression to decrease the immunomodulatory activity.8.The CFSE proliferation experiment results showed that NSGRP promoted the proliferation of the Mouse spleen lymphocytes and the SSGRP inhibited the proliferation as the Control group.Within the concentration of 500-2000?g/mL,SSGRP and NSGRP don't affect the types of Mouse spleen lymphocytes.9.Hoechst 33258 staining assay showd that the SSGRP triggered morphological alteration in RAW 264.7 cells in a dose-dependent manner,however,NSGRP caused few change as the control group.The typical changes including condensation of chromatin,nuclear fragmentation and karyopyknosis were considered as apoptotic.10.Annexin V and PI double-stained apoptosis detection assay showed that the percentage of apoptotic of SSGRP was significantly increased from 15.7%to 30.6%.Our results revealed that sulfur-fumigation significantly inhibited the cellular proliferation of RAW 264.7 cells by inducing apoptosis and S-phase arrest and ??m depolarization collapse.11.The mechanism of apoptosis:The RT-PCR and Western blotting assay were showed that SSGRP promoted macrophages apoptosis through increasing the apoptosis related factor gene and protein expression of Bax and Caspase-8,however,the apoptosis related factors were not activated in the NSGRP group.12.NSGRP up-regulated the expression of JNK and ERK 1/2 protein of RAW 264.7 cells,but through sulfur-fumigation,SSGRP obviously down-regulated the expression of JNK and ERK 1/2 protein,which showed that SSGRP inhibited the MAPK signal pathways.ConclusionThis research mainly revealed that sulfur-fumigation changed the physicochemical property of S.glabra crude polysaccharides and increased the sulfur dioxide residue.SSGRP inhibited the proliferation of the Mouse spleen lymphocytes and promoted macrophages apoptosis,made the non-toxic S.glabra crude polysaccharides to produce certain cytotoxicity,affected the immune system function.Moreover,SSGRP reduced the immunomodulatory activity of the S.glabra crude polysaccharides and the mechanism of sulfur-fumigation was connected to the promotion of apoptosis related factor Bax and Caspase-8 protein expression and the inhibition of the MAPK signal pathways.The study provided a scientific basis and reference to abolish the sulfur-fumigation method.