| Diabetes is a chronic disease characterized by hyperglycemia.Islet β the impairment of cell function is one of the causes.Vitamin B6,also known as pyridoxine,is a water-soluble vitamin that can alleviate diabetes by regulating insulin release and enhancing insulin sensitivity,but its specific mechanism remains to be studied.We used rat islets β cell(RIN-m5F)was used as the research object.By constructing a high glucose cell model,combined with cellular immunofluorescence and Western blot analysis,the effects of different concentrations of vitamin B6 on RIN-m5F cell autophagy were explored.At the same time,the effects of vitamin B6 on RIN-m5F cell apoptosis under high glucose stress were measured by flow cytometry.The effects of different processing conditions on the content of vitamin B6 in egg were determined by high performance liquid chromatography(HPLC).Finally,flow cytometry was used to explore the effect of vitamin B6 in processed egg on RIN-m5F cell apoptosis.The main results are as follows:(1)In order to explore the effect of vitamin B6 on autophagy of RIN-m5F cells,we treated RIN-m5F with 20,40 and 80 μM vitamin B6 under normal or high glucose culture conditions.The changes of autophagy were detected by cellular immunofluorescence and Western blot.The results showed that 20 mM glucose was the best culture condition for high glucose cell model.20 μM vitamin B6 gradually increased the number of autophagosomes in RIN-m5F cells.Further research results show that in the 20 μM vitamin B6 treatment group the ratio of autophagy marker protein microtubule associated protein 1 light chain 3 beta/ microtubule associated protein 1 light chain 3 alpha(LC3-Ⅱ/LC3-Ⅰ)increased significantly,while the protein of sepuestosome 1(SQSTM1/p62)and the phosphorylation level of p70 Ribosomal protein S6 kinase(p70S6K)decreased.The above results showed that vitamin B6 could induce autophagy in RIN-m5F cells under normal and high glucose culture conditions.(2)To analyze whether vitamin B6 induced autophagy can inhibit high glucose induced islets β cells apoptosis,we used flow cytometry to detect the effect of vitamin B6 on RIN-m5F cells apoptosis in high glucose culture.The results showed that 20 mM glucose increased the apoptosis rate from 10.39 % to 22.44 %.20 μM vitamin B6 reduced the apoptosis rate of RIN-m5F cells from 22.44 % to 11.31%.This suggests that vitamin B6 can inhibit high glucose induced islets β Apoptosis.Further studies showed that vitamin B6 reduced the apoptosis rate of RIN-m5F cells from 16.04 % to10.20 %.After treatment with autophagy inhibitor 3-Methyladenine(3-MA),the apoptosis rate of RIN-m5F cells increased from 10.20 % to 23.31 %.These results suggest that vitamin B6 inhibits RIN-m5F cells apoptosis induced by high glucose through autophagy.(3)The content of vitamin B6 in eggs is very high,and eggs are also people’s favorite food.We used high performance liquid chromatography to analyze the changes of vitamin B6 content in eggs under different time and temperature processing conditions and the effect of processing on the biological activity of vitamin B6 in egg yolk.Our results showed that the content of vitamin B6 in egg yolk decreased with the extension of processing time and the increase of processing temperature.The content of vitamin B6 was the highest when the temperature was90 ℃ and the time was 10 min.At the same time,we heated the standard of vitamin B6 at different temperatures and times,and then treated the processed vitamin B6 with islets β cells the apoptosis rate was measured by flow cytometry to explore the effect of thermal processing on vitamin B6 activity.The results showed that vitamin B6 boiled at 100 ℃ for 15 min reduced the apoptosis rate of RIN-m5F cells from23.50 % to 18.82 %,and 3-MA increased the apoptosis rate of RIN-m5F cells from18.82 % to 26.90 %;Vitamin B6 boiled at 90 ℃ for 15 min reduced the apoptosis rate of RIN-m5F cells from 23.50 % to 17.67 %,and 3-MA increased the apoptosis rate of RIN-m5F cells from 17.67 % to 30.50 %;Vitamin B6 boiled at 80 ℃ for 15 min reduced the apoptosis rate of RIN-m5F cells from 23.50 % to 19.45 %;3-MA increased the apoptosis rate of RIN-m5F cells from 19.45 % to 26.31 %;These results showed that thermal processing had no effect on the bioactivity of vitamin B6.Based on the above data,we draw the following conclusions: vitamin B6 inhibits high glucose induced islets through autophagy β cells apoptosis,and hot processing had no effect on the content and activity of vitamin B6 in eggs.Our research clarifies the mechanism of vitamin B6 alleviating diabetes and provides a reference for food processing and people’s healthy diet. |
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