The Study Of Vitamin K2 Promote Differentiation And Mineralization Of MC3T3-E1 Through Autophagy

Objective: vitamin K2 can be used to treat osteoporosis,and it can promote differentiation and fight apoptosis in osteoblasts,but the specific function and mechanism is not clear.Clinically,it is particularly important to the prevention of osteoporosis.in addition to patients with exercise to strengthen bone density,you can also use some drug treatment,such as calcium,Bisphosphonates and so on.vitamin K2 also can be as an auxiliary to prevent osteoporosis,which has attracted more and more attention.At present,autophagy is becoming more and more concerned.In addition to the autophagy effect of tissue cels induced by external stress,the cells also participate in autophagy activities during normal metabolism and differentiation.Because autophagy is closely related with osteoblast differentiation and mineralization.Autophagy is closely connected with osteoporosis disease at the same time,so we can reconsider the effect of prevention osteoporosis on vitamin K2 from the perspective of autophagy.Methods:(1)Mc3t3-e1 osteoblast culture :the mouse cranial apex osteoblast Subclone14(Mc3t3-e1 Subclone14)was derived from the Shanghai cell bank.The cell line was cultured in a medium containing 1% antibiotics,10% fetal bovine serum,and alpha-mem.(2)Osteoblast differentiation of MC3T3-E1: the cels are inoculated in 6-well plate and in each hole cell number is 1 x 104.Three days later,with cell density reaching 70%,we use cell culture medium for osteogenesis differentiation.(3)Making drug treatment groups: fat-soluble vitamin K2 can be dissolved in anhydrous ethanol to make three different concentrations of concentrates,such as 10-2?10-3?10-4M.Finally,using concentrates being diluted respectively to make drug treatment group.Autophagy inhibitor 3-MA and agonist Rapamycin powder reagent are dissolved by PBS to become final concentration(4)Alkaline phosphatase activity assay:using four different concentrations of vitamin k2 to differentiate MC3T3-E1 cels with1,3,5,7 days.The total cell proteins were lysed with an RIPA buffer.And another experimental group was con,3-ma,VK2,VK2+ 3-ma,and the final concentration of autophagy inhibitor(3-ma)was 0.5mm,and the final concentration of vitamin K2 was 10-6m.The protein was extracted for ALP activity detection in 3 days and 5 days.(5)Alizarin red staining of osteoblast differentiation and mineralization,experimental groups can be divided into con,3-MA,VK2,VK2 + 3-MA and con,10-5,10-6,10-7.After differentiation 7 days later,we discarded the supernatant,washing 6-well plate twice by PBS and fixing cels witn 4% paraformaldehyde for 5 mins and finally we go incubation with 0.1% alizarin red staining liquid(6)Western Blot: with the time gradient of 1,3,5,7 days and 0.5 h,1 h,1.5 h,we use the optimal concentration of vitamin K2(10-6 M)to make experiment.extraction time point of protein samples.At the same time,drug group con,Rapamycin,3-ma,VK2,VK2+3-MA were added to the drug treatment when the cell density was about 70%,and the protein was changed every 2 days,and the total cell proteins of the fifth day were lysed with an RIPA buffer(7)immunofluorescence assay: MC3T3-E1 osteoblasts were cultured in 24-well plate,experimental group was con,vk2,vk2 + 3 ma.After culturing 1 h with drugs,the cels were washed twice by PBS and fixed with 4% paraformaldehyde at the room temperature for 15 minutes.Finally added with 0.5% Triton x-100 room temperature for 2 minutes,the cells were blocked by 5% BSA for 2 hours.We use LC3 b antibody to incubate overnight in 4 ? and Alexa Fluor Fluorescence antibody to incubate at room temperature for 1h.Results:(1)vitamin K2 enhanced ALP activity and promoted the differentiation and mineralization of Mc3t3-e1 osteoblasts.When the concentration of vitamin K2 was 1u M(10-6m),the ALP activity was significantly enhanced(P<0.05),while the other concentration was inhibited.Compared with the control group,ALP activity was obviously the strongest in the 5th day(p < 0.01).When the concentration of vitamin K2 was 1u M(10-6m),the number of red mineralized nodules increased significantly.(2)vitamin K2 stimulates Mc3t3-e1 osteoblasts to produce autophagy.Vitamin k2(10-6 M)stimulates MC3T3 E1 osteoblast at various time points of 1,3,5,7 days and ranging from 0.5 h to 0.5 h to generate different conversion rates of LC3 I.The results show that conversion rates of LC3 I were increased at 0.5 h,1 h,1.5 all time points compared with control group.In the first hour of 1,3,5,7 each day,the difference was significant compared with the control group(p < 0.05).(3)vitamin K2 makes the Mc3t3-e1 cells produce the strongest ALP(day 5),LC3 protein expression in this time:The conversion rate of LC3II/lc3 I was increased at 0.5h and 1h(P<0.05),and the conversion rate of LC3II/lc3 I was significantly increased at 1.5h(P<0.01).(4)vitamin K2 stimulates Mc3t3-e1 osteoblasts to produce autophagy in immunofluorescence: The amount of fluorescence produced by the vitamin K2 group was higher,and the number of fluorescent small bodies produced by the autophagy inhibitor 3-ma group was smaller.(5)autophagy inhibitor 3-ma inhibits osteogenesis differentiation and mineralization.The conversion rate of LC3II/LC3 I was significantly reduced(P<0.05)compared with that of the vitamin k2 drug treatment group.Compared with the control group,the 3-ma group had significant inhibitory effect on ALP,with significant difference(P<0.05),and the v K2+ 3-ma group had significant inhibitory effect compared with the v K2 group(P<0.05).Conclusion :(1)vitamin K2 can promote the mineralization of Mc3t3-e1 osteoblasts.(2)vitamin K2 is used in osteoblasts Mc3t3-e1 to produce autophagy?(3)the autophagy produced by vitamin K2 promotes the mineralization of osteoblasts Mc3t3-e1.