Study On The Protective Effect Of Curcumin On BMSCs Under High Glucose And Serum-free Environment And Its Mechanism Of Promoting Wound Healing

Objective:In recent years,the incidence of diabetes has risen sharply.Due to long-term poor blood glucose control,angiogenesis and blood perfusion disorders have led to the occurrence of ischemic ulcers and gangrene on the foot,forming chronic wounds,which has brought challenges to treatment.With the continuous development of biotechnology,stem cell transplantation has become a new way to treat lower limb ischemic diseases and diabetic wounds,bringing hope for treatment.Therefore,this study took the local microenvironmental change of the diabetic wound as the starting point,established a high-glucose and serum-free environment in vitro to simulate the state of high-glucose ischemia in diabetic patients.Under the high-glucose and serum-free environment,we preliminarily explored the effect of curcumin on the bone marrow.The influence of bone marrow-derived mesenchymal stem cells(BMSCs).First,we preliminarily verified the effect of curcumin on the apoptosis rate and antioxidant capacity of BMSCs by culturing BMSCs in a high-sugar and serum-free environment.Secondly,under the environment of high glucose and serum-free cells in vitro,further study the effect of curcumin on the paracrine and migration ability of BMSCs and preliminary explore its mechanism of action.Methods:(1)BMSCs were extracted from Sprague-Dawley(SD)rats and subcultured in vitro.When the cells were cultured to the third generation(Passage3,P3),they were used to identify their surface molecular antigens by flow cytometry.Osteogenic staining to detect cell differentiation potential.(2)Take the third to fourth generation BMSCs with good growth status and culture them overnight.Use the CCK8 method to investigate the effect of curcumin on the survival rate of bone marrow mesenchymal stem cells.Use this step to determine the safe concentration of curcumin for subsequent use experiment.After the safe drug concentration of curcumin was determined,the third generation of BMSCs were taken,and BMSCs were cultured in a high-sugar and serumfree environment for 48 hours to simulate the high-sugar-ischemic environment of diabetic wounds.The experiment was divided into 5 groups according to the following conditions,namely the blank control group,Model control group,and curcumin drug group with different concentrations(0,1,5,10μmol/L),blank control group BMSCs culture condition is DMEM/F12 medium containing 5.5mM glucose,10% serum concentration,drug group and high The sugar-free serum model group was consistent,all were cultured in 33 mM glucose,serumfree DMEM/F12 medium;48h later,the morphological changes of BMSCs were observed,and the apoptosis level and intracellular reactive oxygen species(ROS)content of each group were detected;use ELISA method was used to detect the expression of superoxide dismutase(SOD),catalase(chloramphenicol acetyltransferase,CAT)and malondialdehyde(MDA)in each group of cell oxidative stress indicators under high glucose and serum-free environment.(3)According to the same experiment as above,the cells were cultured for 48 hours,and then the BMSCs were scratched,and the relative cell migration rates of the blank control group,model group and drug group were compared at 0h and 24h;qPCR detection of each group of paracrine factors: cell transforming growth factor(TGF-β),vascular endothelial growth factor(VEGF),fibroblast growth factor2(FGF2),platelet growth factor(PDGF)The mRNA expression level was detected by Western blot(WB)to detect the expression of matrix metalloproteinase(MMP2)protein in each group of cells.(3)According to the same experiment group as above,after the cells were cultured for 48 h,the BMSCs were scratched and photographed under the microscope at 0h and 24h.Using the microscope matching measuring tool,the width of the cell scratch was measured,and the relative cell migration rate was calculated;real-time fluorescence was adopted.QPCR detection of m RNA expression levels of TGF-β,FGF2,PDGF,and VEGF.WB to detect the expression of MMP2 protein in each group of cells.Results:(1)The extracted rat BMSCs cells showed spindle-shaped and fibrous growth.The surface markers of BMSCs CD90 and CD29 were positive by flow cytometry,and CD45 was negative.The cells have the potential for osteogenic and adipogenic differentiation.(2)It can be concluded from the results of CCK8 that curcumin below20μmol/L has no obvious toxic effect on cell proliferation(P>0.05);compared with the high glucose and serum-free model group,curcumin 10μmol/L can be seen under the microscope Group BMSCs decreased floating cells and improved cell morphology;flow cytometry results showed that the apoptotic rate of cells in a high-glucose and serum-free environment increased(P<0.01).After treatment with different concentrations of curcumin(1,5,10μmol/L),The apoptotic rate gradually decreased.In the 1μmol/L group,the apoptotic rate was not much different from the model control group(P<0.01).The 5 and 10μmol/L groups could significantly improve the apoptosis level(P<0.05);inverted fluorescence Microscopic examination results showed that compared with the blank control group,BMSCs cultured with high glucose and serum-free showed obvious green fluorescence under an inverted fluorescence microscope,while after treatment with low,medium,and high curcumin drugs,the cell fluorescence intensity gradually decreased.In the 1μmol/L group,compared with the high-glycemic serumfree model control group,the intracellular fluorescence intensity was not much different(P>0.05).In the 5 and 10μmol/L groups,the intracellular fluorescence intensity decreased(P<0.05);You Liu The results of the cytometer test showed that the expression of intracellular ROS in the high glucose and serum-free environment was significantly higher than that of the blank control group(P>0.01).After different concentration gradients of curcumin drugs were used,it was compared with the high-glycemic serum-free model control group.In contrast,when the concentration of curcumin was 10μmol/L,there was a significant difference in the reduction of intracellular ROS(P<0.01);the oxidative stress index test showed that compared with the blank control group,the intracellular ROS decreased under high glucose and serum-free environment.The expression of SOD decreased(P<0.01),the expression of CAT decreased(P<0.05),and the content of MDA increased(P<0.01).Compared with the model control,after giving different concentrations of curcumin,the expression of SOD in cells increased.Concentrations of 5 and 10μmol/L are statistically different(P<0.05);intracellular CAT expression increases,with curcumin concentration at 10μmol/L,there is a statistical difference(P<0.05);intracellular MDA content decreases,curcumin concentration 5μmol/L is statistically different(P<0.05),when the curcumin concentration is 10μmol/,there is a significant difference(P<0.01).(3)The results of the cell scratch test showed that compared with the blank control group,the migration ability of BMSCs was significantly weakened in the high-sugar serum-free model group 24 hours after scratching(P<0.01),while the cells migrated after curcumin was given.The rate has increased.Curcumin concentration of 10μmol/L can significantly increase the migration rate of BMSCs(P<0.01);qPCR test data show that the m RNA expression of TGF-β,FGF2,PDGF,and VEGF is significantly down-regulated in a high glucose and serumfree environment(P<0.01),under the action of different concentrations of curcumin drugs,the m RNA expression levels of TGF-β,FGF2,PDGF,and VEGF increased in a high glucose and serum-free environment,and curcumin concentrations of 5 and 10 μmol/L have a significant effect(P<0.01);WB results showed that the expression of MMP2 in the model control group was significantly lower than that in the blank control group(P<0.01),and after treatment with curcumin drugs,the MMP2 protein increased,and the concentration of curcumin was 10μmol/L.Sexual difference(P<0.01).Conclusion:(1)The SD rat BMSCs were successfully extracted,and the cell surface antigen phenotype test met the international standards,showing the potential for proliferation and multidirectional differentiation.(2)Curcumin concentration below 20μmol/L has no toxic effect on cells;high glucose and serum-free environment will obviously damage BMSCs,curcumin has a protective effect on BMSCs under high glucose environment(inhibition of intracellular ROS production is related to,regulating The expression of the antioxidant enzymes SOD and CAT in the cell reduces the level of MDA),thereby resisting the production of apoptosis and oxidative stress damage of BMSCs.(3)Curcumin can up-regulate the expression of paracrine-related factors VEGF,TGF-β,PDGF and FGF2 in BMSCs in a high-glucose serum-free environment,thereby improving the paracrine ability of BMSCs in a high-glucose serum-free environment;curcumin promotes elevation The mechanism of cell migration in sugar-free serum is related to the up-regulation of paracrine-related genes and the increase of MMP2 protein expression.