Effects Of Differential Expression Saliva Proteins On OLP Patients With Huashi Xingyu Qingre Decoction Treatment

Objective: Oral lichen planus(OLP) is a chronic oral mucosal disease under the influence of immune factors, infection factors, psychological and other relevant factors. The World Health Organization(WHO) enrolled this disease as Precancerous condition because it's stubborn refractory and easy to become cancer. The main clinical manifestations is white damage and pain, and the severe cases can affect the patient's quality of life. Therefore, the early diagnosis and treatment for OLP is particularly important. So far, the domestic and foreign scholars put forward many methods for the treatment of OLP, mainly including medication, surgery treatment and physical therapy, etc. But western medicine were used to improve symptoms manly on the application of corticosteroids or immune regulator to local and systemic, which were often discontinued due to appear more serious side effects. Recently, the Chinese medicine treatment of OLP is paid to more attention and shows the superiority. In clinic, we found Huashi Xingyu Qingre decoction therapy has better clinical curative effect, but the exact paraphrase mechanism is not very clear. In this study, the differentially expressed saliva proteins of OLP before and after treatment were screened by two-dimensional fluorescence difference gel electrophoresis and identified by liquid chromatography-mass spectrometry. The changes of the differences in protein are further detected by Western-blot which provides the theory basis for the possible pathogenesis of OLP and the treatment of traditional Chinese medicine.Methods:1 The differentially expressed saliva proteins before and after treatment were screened by two-dimensional fluorescence difference gel electrophoresis and identified by liquid chromatography-mass spectrometry.By Huashi Xingyu Qingre decoction treatment of OLP prescriptions eight weeks, six cases of OLP patients before and after treatment were selected and saliva sample was collected. Total saliva proteins were cleaned by the 2-D Clean-Up Kit and protein concentration was determined by Bradford method to insure sample volumes accurately. Saliva proteins were separated by two-dimensional fluorescence difference gel electrophoresis, then the electrophoresis pattern can be obtained by Typhoon9410. The different proteins which were greater than 1.2 were found and labeled by Decyder TM2D6.5 software. The differentially expressed saliva proteins can be picked by Ettan Picker in gel and identified by liquid chromatography-mass spectrometry(LC-MS).2 The expression of the saliva proteins were detected by Western-blot technique.Saliva proteins of thirty OLP patients before and after Huashi Xingyu Qingre decoction treatment was extracted.To validate and further test the effects of treatment to differential proteins content, Secretory IgA, IgM, serum albumin, carbonic anhydrase and Zinc ?-2-glycoprotein were detected by western blotting technique. The saliva proteins were separated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). The protein bands were transferred by half dry transfer method. The purpose proteins were scanned and exposed in Alpha innotech after the protein antibody incubation to obtain the stripe image of target proteins. Quantity One was used to analyze the grey value of the purpose protein bands. Database was analyzed by SPSS13.0.Results:1 2-D DIGE: In the 2-D DIGE image, a number of protein spots were overlapped by Cy3 and Cy5 two fluorescent colors, presenting yellow. This showed the proteins of OLP patients before and after treatment were at the same levels of expression. While some highly expression of specific proteins presenting red or green showed that OLP patients before and after treatment were at the different levels of expression. The highly expression spots analyzed and labeled by differential analysis software version 6.5 and liquid chromatography-mass spectrometry showed that they were concurrence but expressed different. The differentially expressed saliva proteins were searched by Bioworks Browser 3.3.1: Secretory IgA, IgM, serum albumin, carbonic anhydrase, Zinc ?-2-glycoprotein, salivary amylase;2 Western-blot: Saliva proteins were separated by SDS-PAGE. The electrophoresis patterns were clear. Proteins loss and degradation were rarely to be found. The images scanned by Alpha innotech showed that the purpose protein bands were clear. Five differential proteins was detected with different intensity before and after treatment. The expression of IgM, carbonic anhydrase, Zinc ?-2-glycoprotein and serum albumin after treatment were significantly lower than those before treatment, while Secretory IgA were higher. The differences were statistically significant.Conclusions:1 The highly expression spots of OLP selected and identified by two-dimensional fluorescence difference gel electrophoresis and liquid chromatography-mass spectrometry showed that the differentially expressed saliva proteins are Secretory IgA, IgM, serum albumin, carbonic anhydrase, Zinc ?-2-glycoprotein, salivary amylase;2 After Huashi Xingyu Qingre decoction treatment, the expression level of IgM, carbonic anhydrase, Zinc ?-2-glycoprotein and serum albumin in OLP were decreased, while Secretory IgA increased. Secretory IgA, carbonic anhydrase, Zinc ?-2-glycoprotein, serum albumin and IgM may play a vital part on the occurrence and development of oral lichen planus.3 Secretory IgA is one differentially expressed saliva protein which expresses stably and good repeatability, so it can be considered as biomarkers in the study about OLP patients saliva protein.