Improving Effect Of Cinnamic Aldehyde On Fibroblasts Migration Under Hyperglycaemic Condition And Its Underlying Mechanism

BackgroundDiabetes ulcers(Diabetes ulcers,DUs)have a high incidence and recurrence rate,and which has become a major factor in non-traumatic amputation for its complex abnormal pathophysiology and difficulty to heal.DUs seriously affect the life quality of patients because of the leading role in non-traumatic amputation and delay of healing process,which also increase the morbidity,mortality and medical costs of patients.However,the molecular and genetic mechanisms of the pathogenesis of DUs are not clear.Therefore,to further explore the underlying mechanism and effective treatment regimen is an urgent issue to be solved.Recent studies have shown that the sustained hyperglycemia contributes to increasing the level of reactive oxygen species(ROS)and exacerbating oxidative stress(OS)damage in the body,which is one of the main causes that leads to the delay of diabetic wound healing.Nuclear factor erythroid2-related factor2(NRF2)is a redox-sensitive transcription factor that can neutralize the excessive ROS and keep the balance of redox homeostasis through regulating the expression of dozens of antioxidant enzymes and phase ? detoxification enzymes downstream.Hence,NRF2 has become a new therapeutic target for DUs.Our study group have devoted to researching the NRF2 field for a long time.We have found that NRF2 plays an essential role in diabetic nephropathy and diabetic skeletal muscle lesions.Recently,our findings have confirmed that the skins are under serious oxidative stress damage at the wound site of DUs,and pharmacological activation of NRF2 pathway significantly improve diabetic wound healing.As we all known,cinnamon whose active ingredients is cinnamon aldehyde(cinnamicaldehyde,CA),has been widely used as a traditional Chinese herb or seasonings.So far,a number of studies have verified that CA has a wide range of biological and pharmacological effects.Our recent research has demonstrated that CA,as a specific NRF2 agonist,significantly promotes keratinocytes migration,alleviates oxidative stress damage and improves STZ-induced diabetic wound healing.Based on the results of the previous studies,we aimed to further explore whether CA could modulate the migration and proliferation of fibroblast which is another essential cell type involved in wound healing through activating the NRF2 pathway.Therefore,to provide experimental evidence indicating that CA could be used therapeutically to improve DUs,the current study observed the effect of CA and explored the role of NRF2 on the function of fibroblasts(HS27)under high glucose condition.Research purposes1.To observe the effect of CA on migration of fibroblasts under hyperglycemia condition.2.To explore the role of NRF2 pathway in migration of fibroblasts.3.To explore the underlying mechanism of CA on migration of fibroblasts.Materials and MethodsExperimental Materials:Human skin fibroblast cell line HS27 was given from Dr.Tim Bowden,Arizona Cancer Center;CA was purchased from Sigma.Groups of experiments:The study includes normal glucose(NG,5.5mmol/L)group,the high glucose(HG,30mmol/L)group,(HG+ConsiRNA)group and the(HG+NRF2siRNA)group.The concentration of CA is 20?mol / L.Experimental methods:1.Wound healing assay was used to observe the effect of CA or blocking the NRF2 pathway on the migration of HS27 cells under hyperglycaemic conditionTo create an empty space for cell migration in the border,a polydimethylsiloxane(PDMS)barrier in a 6-well plate for wound healing assays was utilized before the experiment.HS27 cells were cultured in NG or HG for 2 days.After a confluent cell monolayer was formed,the slab was removed and cells were allowed to migrate for 72 h.The HG group was treated with CA(20?mol/L)when seeding them and retreated every 24 hours until the end of this experiment.The cell migration rates were evaluated by taking pictures at indicated time 0h,24 h,48h,60 h,72h.For siRNA experiments,the cells were seeded into 6-well plates and transfected with Consi RNA and NRF2 siRNA respectively,a confluent cell monolayer was formed after 48 hours and the slabs were removed and observed as described above.2.Immunoblot analysis was performed to detect the effect of CA or blocked the NRF2 pathway on the level of proliferation and apoptosis related proteins expressed in fibroblasts.The cells were divided into four groups: HG,(HG+CA),(HG+ConsiRNA),(HG+NRF2si RNA).After 48 h,the cell proteins were extracted and analyzed the expression levels of NRF2,HO-1,P53 and P21 by immunoblot.3.ROS were measured in HS27 cells treated with CA or transfected with NRF2 si RNA using 2`,7`-dichlorofluorescein diacetate(DCF).The cells were seeded into six groups: negative control,positive control,NG,HG,(HG+CA),(HG+Consi RNA),(HG+NRF2siRNA).After cultured for 48 h,the cells were washed with HBSS twice and switched into fresh HBSS containing 10 ?g/mL DCF-DA,incubated for 30 min.Then,the cells were washed 3 times with HBSS and the fluorescence intensity was measured by fluorescence microscope(Zeiss Observer.Z1)with excitation and emission wavelengths of 485 nm and 530 nm.Fluorescent images were analyzed with Slidebook 5.0 software.The relative fluorescence intensity,which represents the amount of intracellular ROS from 3 random fields,was analyzed using Image J software.Research result1.CA significantly promote the migration of HS27 cells under high glucose conditions.There was no difference in migration of HS27 cells among NG group,HG group and(HG+CA)group for the first 24 h.However,compared to NG group,the migration of HS27 cells in HG group was significantly blocked at 48 h,and the migration of HS27 cells in(HG+CA)group was significantly improved when treated with CA.At 60 h,the migration in HG group were still slower than NG group.The fibroblasts of NG group were completely healed till 72 h,and so did the(HG+CA)group,while the wound closure rate was only 70% in HG group.Furthermore,in order to explored whether NRF2 was involved in CA promote migration of fibroblasts,the NRF2 siRNA was transfected into fibroblasts and observed the migration between the(HG+NRF2si RNA)and(HG+ConsiRNA)groups.There was no difference at the first 48 h.While,the wound closure rate of(HG+NRF2siRNA)group was significantly lower than(HG+ConsiRNA)group.And,at 72 h,the wound of(HG+Consi RNA)group was completely closed,however,there was still 24% of the wound area haven't closed in(HG+NRF2si RNA)group.These results suggest that blocking NRF2 pathway inhibit the migration of fibroblasts.2.CA activates the NRF2 pathway,but does not affect the expression of P21 and P53 which related to cell proliferation and apoptosis.After treatment with CA,the mobility has been significantly improved and the expression of NRF2 and HO-1 were notably increased,while the expression of P21 and P53 which were associated with proliferation and apoptosis were unchanged between the two groups.These results indicated that CA activates the NRF2 signaling,but have no effect on cell proliferation and apoptosis.Moreover,the expression of proliferation and apoptosis related proteins P21 and P53 were measured after transfected with NRF2 si RNA.The results have shown that knocking down of NRF2 and its downstream HO-1 in(HG+NRF2si RNA)group did not affect the proliferation and apoptosis of HS27 cells.These results showed that the NRF2 pathway didn't affect the expression of proliferation and apoptosis associated proteins P21 and P53 either.3.CA alleviated hyperglycemia-induced ROS production in HS27 cells.The ROS level were measured among NG group,HG group and(HG+CA)group.The results have showd that the ROS levels was clearly increased in HG group than the NG group,and the CA obviously reduced the ROS level induced by hyperglycemia in HS27 cells.However,as the ROS measurement between the(HG+NRF2siRNA)group and(HG+Consi RNA)group showed that knocking down of NRF2 far more increased intracellular ROS levels in HS27 cells.These results suggested that the NRF2 pathway play an essential role in modulating the level of hyperglycemia-induced ROS.Conclusions1.The migration ability of fibroblasts is evidently impaired under hyperglycemia condition;and CA aleviates the inhibitory effect of high glucose on the migration of fibroblasts;to block the NRF2 pathway in high glucose,the inhibitory effect of high glucose is further aggravated.These results suggest that CA promotes the migration of fibroblasts under hyperglycaemic conditions,and NRF2 pathway may play an important regulatory role in the migration of fibroblasts.2.Cinnamaldehyde activates NRF2 pathway in fibroblasts under hyperglycaemic condition,but does not affect the expression of proliferation and apoptosis-related proteins P21 and P53.3.High glucose notably increased the level of ROS in HS27 cells,CA could reduce the level of ROS induced by high glucose.Furthermore,the levels of ROS in fibroblasts was far more increased after transfected with NRF2 siRNA.These results indicate that CA can reduce ROS levels induced by high glucose in fibroblasts by NRF2 pathway.Collectively,our findings indicated that CA significantly promote the migration of fibroblasts under hyperglycaemic condition.Although it did not affect the expression levels of proliferation and apoptosis related proteins in fibroblasts,CA upregulates the levels of NRF2 and its downstream HO-1,and CA distinctly reduces hyperglycemia-induced ROS production in HS27 cells.Therefore,CA promotes the migration of fibroblasts under hyperglycaemic condition,and its mechanism may be related to the activation of NRF2 pathway and the reduction of oxidative stress damage.