Effects And Mechanism Of Trilobatin On Wound Healing In Rats

Objective: To investigate the effects of trilobatin(TLB)on wound healing in rats and its possible mechanism.Methods: Male Sprague-Dawley rats(170-200 g)were divided into four groups by simple randomization: model group(Model),model + TLB 0.05 g/kg group(Model + TLB0.05 g/kg),model + TLB 0.25 g/kg(Model + TLB 0.25 g/kg),and model + TLB 1.25 g/kg(Model + TLB 1.25 g/kg).A circular wound with a radius of 1 cm was prepared by excising the full thickness of the skin with a scalpel at 2 cm of the subscapular angle in the midline of the back of the rats.One gram of TLB ointment was evenly applied to the wound twice a day for 14 days.Before each treatment,the wound healing was observed and photographed.The wound was photographed on the day of 0,3,7,10 and 14 after model establishment at a fixed height and angle by camera.The wound healing area was converted and statistically analyzed by Image J image analysis software.After administration for 14 days,the local tissue cell infiltration in the wound of rats in each group was observed by hematoxylin-eosin(H&E)staining;the collagen deposition in the wound tissue was observed by Masson staining;the number of new blood vessels in the wound tissue of rats was detected by immunohistochemical(IHC)method.Enzyme-linked immunosorbent assay and immunofluorescence were used to detect the levels of inflammatory cytokines TNF-α and IL-1β in rats serum and the protein expression of TNF-α and IL-1β in wound tissue,respectively;Western blot was used to detect the protein expression of NF-κB(p65),NF-κB(p50),Akt and their phosphorylation levels,VEGFA,VEGFR-2,MMP9,MMP2 and TIMP1.Results: The results showed that the wound healing rate of TLB-treated group(91.3%,93.7%,95%)significantly higher than Model group(86.6%).The number of new capillaries,fibroblasts,and epidermal growth thickness in the wound tissue of TLB groups were markedly increased.In addition,TLB obviously reduced scar area in local skin tissue,alleviated inflammatory cell infiltration,and dramatically promoted collagen deposition and neocapillarization.Compared with TNF-α(238.9 pg/m L)and IL-1β(41.6 pg/m L)in the serum of rats in Model group,TLB(medium and high doses)notably decreased TNF-α(207.1 pg/m L,192.8 pg/m L)levels,while TLB(low,medium,and high doses)significantly reduced IL-1β(37.7 pg/m L,35.8 pg/m L,34.2 pg/m L)in rats serum.The positive area of TNF-α and IL-1β in the wound tissue of Model group were markedly increased,while after TLB treatment the expression area of TNF-α and IL-1β were significantly lower than those of Model group.Compared with Model group TLB medium and high dose groups not only obviously down-regulated the expression of MMP2 and MMP9 in wound tissue,also remarkably decreased the phosphorylation levels of NF-κB(p65)and NF-κB(p50).TLB could increased the phosphorylation level of Akt and up-regulated VEGFA and VEGFR-2 as well as TIMP1 protein expression in wound tissue.Conclusion: TLB promotes wound healing and its mechanism may be related to the regulation of MMP9,MMP2/TIMP1 balance,inhibition of pro-inflammatory release,and promotion of collagen deposition and angiogenesis.