| Objective The submandibular gland cells of patients with Sjogren's syndrome were used as subjects,and the control mechanism of the disease-related protein channel was established with Strengthening Qi,Nourishing Yin and Removing Blood Stasis FormuIa.lt will provide a wider range of ideas for the clinical treatnent of SS.Methods 1.Effects of different concentration ratio of Yiqi Yangyin Quyu Recipe on expression of AQP5 and M3R protein in submandibular gland cells:?Strengthening Qi,Nourishing Yin and Removing Blood Stasis Formula drug containing serum was prepared by SD rats,set the 5%medicated serum group,10%of medicated serum group,15%of medicated serum group,20%of medicated serum group,30%of medicated serum group,blank serum,serum-free group and blank group,a total of eight groups,and take five time points as the longitudinal comparison,with CCK-8 method to detect different concentration ratio of Yiqi Yangyin Quyu Recipe side effect on the activity of submandibular gland cells.CDRespectively,set up the following groups:blank group(only contains RPMI1640 culture),10%of the proportion of blank serum with RPMI1640 group,and set to contain respectively 5%,10%,15%,20%,different concentrations of medicated serum with RPMI1640 culture group,a total of six groups.After cultured for 24 hours,the expression of AQP5 and M3R protein was detected by Western-blot.?Set the blank serum group,5%medicated serum group,10%medicated serum group,30%medicated serum group,a total of four groups,with laser confocal observation.2.Effects of Yiqi Yangyin Quyu Recipe,interferon gamma,interferon gamma antagonists on expression of AQP5,M3R and flotillin-2 in Submandibular Gland of Sjogren's syndrome:Set to six groups of the cultured cells,were given the following processing:blank group(only contains RPMI1640 culture),blank serum group(10%of the proportion of blank serum with RPMI1640 culture),drug containing serum group(10%of the Yiqi Yangyin Quyu Recipe drug-containing serum with RPMI1640 culture),interferon gamma group(IFN-? with RPMI1640 culture medium),interferon gamma with interferon gamma antagonist group(IFN-? and IFN-? antagonist with RPMI1640 culture medium),interferon gamma with Yiqi Yangyin Quyu Recipe drug-containing serum group(IFN-? and 10%of the Yiqi Yangyin Quyu recipe serum with RPMI1640 culture medium),a total of six groups,cultured cells for 24h.? The expression of AQP5 and M3R protein in submandibular gland cells were detected by Western-blot.? The expression of AQP5 mRNA and M3R mRNA in submandibular gland cells were detected by RT?PCR.? The expression of AQP5 and flotillin-2 protein in submandibular gland cells were observed by laser confocal microscopy.Results 1.The effects of different concentrations of Yiqi Yangyin Quyu Decoction on the expression of AQP5 and M3R protein in submandibular gland cells of SS patients were investigated.?Cell viability(OD)was measured:The OD value of 10%serum group was higher than that of blank serum group and serum-free group when the cells were cultured for 12 hours,(P<0.05),the difference was statistically significant.The OD value of 10%,15%,20%and 30%drug-containing serum group was higher than that of serum-free group,and the OD value of 10%and 15%drug-containing serum group was higher than that of blank serum group when the cells were cultured for 24 hours,(P<0.05),the difference was statistically significant.The OD value of 10%,15%,20%and 30%drug containing serum was higher than that of serum-free group,and the OD value of 10%,15%,20%drug-containing serum group was also higher than that of the blank serum group,(P<0.05),the difference was statistically significant.But there was no significant difference between the 30%drug containing serum group and the blank serum group,(P>0.05).The OD value of 10%and 15%medicated serum group was not only higher than that of serum-free group,but also than blank serum group when the cells were cultured for 72 hours,C(P<0.05),the difference was statistically significant.?AQP5,M3R protein expression assay:The 5%medicated serum group is higher than the blank group of AQP5 protein expression,10%medicated serum group is higher than 5%medicated serum group of AQP5 protein expression,15%medicated serum group is higher than the blank group,and also higher than 5%medicated serum group and 10%medicated serum group(P<0.05),the difference was statistically significant.M3R protein expression of the same trend is consistent with the AQP5.The 15%medicated serum group was higher than 10%medicated serum group and s%medicated serum group and blank serum group(P<0.05),the difference was statistically significant,but not different with 20%medicated serum group(P>0.05),the difference was not statistically significant.(3)Laser confocal microscopy:Intracellular AQP5,M3R fluorescence intensity enhancement.The AQP5 labeled in the 10%serum group was significantly higher than that in the blank serum group,5%medicated serum group and 30%medicated serum group.The 5%medicated serum group of M3R expression was higher than that of the blank serum group,but weaker than the 10%serum group.2.Effects of Yiqi Yangyin QuyU Recipe,interferon gamma,interferon gamma antagonists on expression of AQP5,M3R and flotillin-2 in submandibular gland of Sjogren's syndrome:CDAQP5,M3R protein expression:Westem-blotting was used to detect the expression of AQP5 and M3R in interferon gamma group,which was lower than that in interferon gamma with antagonist group and interferon gamma with Yiqi Yangyin Quyu Recip group(P<0.05).The latter two comparison(P>0.05),there was no statistically significant difference·?AQP5 mRNA,M3R mRNA expression:The expression of AQP5 mRNA and M3R mRNA in interferon gamma with antagonist group was significantly higher than that in interferon gamma group(P<0.05),but there was no significant difference with interferon gamma with Yiqi Yangyin Quyu Recipe grouP,(P>0.05).Similarly,the expression of AQP5 mRNA and M3R mRNA in interferon gamma with Yiqi Yangyin Quyu group was higher than that in interferon gamma group,(P<0.05),the difference was statistically signifieant.?Laser confocal microscopy:The expression of AQP5 protein was increased after Yiqi Yangyin Quyu Recipe interfered with the submandibular gland cells.And after treatment of submandibular gland cells by IFN-?,the fluorescence intensity of AQP5 in cells was significantly decreased.The expression of AQP5 in interferon gamma antagonist group was higher than that in interferon gamma group,and the expression position of AQP5 has changed.Conclusion:Yiqi Yangyin Quyu Recipe has the effect of regulating the distribution of aquaporin AQP5 and regulating the expression of M3R in submandibular gland cells of SS patients,so as to control the water transport in salivary glands.In the stimulation of interferon gamma,submandibular gland cells in the expression of AQP5 greatly reduced,so this is likely to be one of the important factors in the pathogenesis of SS.Interferon gamna antagonist and Yiqi Yangyin Quyu Recipe ean effectively prevent the problem which was the interferon gamma induced the AQP5 abnormal distribution in submandibular gland cells,then maintain normal water transport.lt can be used as an important basis for the treatment of SS. |
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