| It is estimated that more than 400 million people worldwide have the wound caused by different causes, it was found that the use of antibiotics during the treatment of wounds, prone to resistance, and caused injury to tissue, prolonged the healing time. When the moist dressing appeared, the researchers at home and abroad used honey smear on the healing of wound and obtained the good effect, and the vast majority report of honey to treat the wound sure honey effect and application value.However, the current study on honey promote wound healing mechanism is still unclear, needs further research. Study in this paper will use honey and highest level phenolic acid composition in honey as the research object, through the establishment of animal trauma model, observation of honey and phenolic acids in wound tissue morphology change and related growth factor influence, honey and phenolic acids were discussed from cellular and molecular level of trauma repair skin, help to strengthen domestic honey and phenolic acids material used in trauma care of basic research and clinical research, and provide reference for clinicians to choose honey dressing raw material source, has very important practical significance.Objective:To establish the rat model of skin wound and observe the influences of honey and its three phenolic acid composition on wound pathological changes and related cytokines.Methods:180 Wistar rats were randomly divided into 12 groups, namely blank group, untreated group, honey group,2 times GA(gallic acid) group,1 times GA group,0.5 times GA group,2 times PA(p-coumarin acid) group,1 times PA group,0.5 times PA group,2 times CA(caffeic acid) group,1 times CA group,0.5 times CA group(animal number:4/16/16/16/16/16/16/16/16/16/16/16). The skin wound were made by 6mm bio punch except the blank group. Hematoxylin and eosin(HE) staining pathological section of skin wound tissue was made to observe the pathological changes.The content of serum fibroblast growth factor 2(FGF2) and transfer growth factor β1(TGFβ1) were measured by enzyme linked immunosorbent assay(ELISA) on 1, 3,5,7 days after medication.Results:(1) The scores of inflammation infiltration, collagenous fiber proliferation score and blood capillary proliferation in skin wound HE staining: 1) Inflammation infiltration:①Blank group and untreated group:There were no inflammation inflitration changes in each time point in blank group and the score were 0. In untreated group, appeared at first day, third day rised to peak, then stayed the declining trend at 5th day and 7th day. Prompt, in the process of the normal healing of the wound, after rising first less inflammatory infiltrates.②Honey group on first day rised to the peak and higher than untreated group which reached the peak on third day. The 5th and 7th day, honey group and untreated group were in decline, but untreated group was still higher than honey group, and honey declined on the 7th day fell to blank group and levels. The results indicated that honey showed pro-inflammatatory responses which led to increase inflammatory responses in early stage and anti-inflammatory responses which led to decrease inflammatory responses in late stage, reduced the inflammatory time.③Phenolic acid groups:On first day, inflammatory infiltration appeared and rised to peak on first day and third day. Phenolic acid groups whose peak appeared on first day declined on third day and the peak score were lower than honey group, and higher than untreated group whose pesk hadn’t arised. This showed that pro-inflammatatory responses in phenolic acid groups were stronger than untreated group but weaker than honey group. Phenolic acid groups whose peak appeared on third day were higher than honey group which were in decline, lower than untreated group whose peak arised at this time. This showed that pro-inflammatatory responses in phenolic acid groups whose peak arised on third day were weaker than honey group. Then on the 5th day and on the 7th day all phenolic acid groups were indclined, and the scores were higher than honey group but lower than untreated group. This showed that anti-inflammatatory responses in phenolic acid groups were stronger than untreated group but weaker than honey group. And showed that pro-inflammatatory and anti-inflammatatory responses in phenolic acid groups were stronger than untreated group but weaker than honey group.④Pifferent times dose in same phenolic acids:Different dose GA group except the first day,2 times group higher than 1 times group which higher than 0.5 times group, all showed increased and decreased in different degrees and showed the same level on third day, then stayed decline trendency and the same score. This showed that pro-inflammatory response in 2 times group was the best,1 times the second and 0.5 times the last; there were no difference on anti-inflammatory responses in different dose of GA groups. Different dose of PA groups and CA groups stayed the same changes trendency, showed that inflammatory infiltration didn’t relate with phenolic acid dose.⑤Different kinds of phenolic acids in the same times between groups are compared: both 2 times groups,1 times groups or 0.5 times groups between the different kinds of phenolic acids were compared, GA group inflammatory infiltration score at first day were the highest (P<0.05). This indicated that pro-inflammatatory responses in GA was the best; however, same times dose groups after third,5th,7th day in varying degrees of increased or decreased, the GA group in three groups of phenolic acids were in the lowest position (P<0.05). This indicated that anti-inflammatatory responses of GA groups were the strongest in three phenolic acids groups. This indicated that GA were the best.2) Collagenous fiber proliferation and blood capillary proliferation score:①Blank group and untreated group:There were no collagenous fiber proliferation and blood capillary proliferation at first day and the socres were 0.Untreated group rised on the third,5th and 7th day, and peaked on 7th day. This indicated that in natural state, collagenous fiber proliferation and blood capillary proliferation rised on third day.②Honey group:There were no collagenous fiber proliferation and blood capillary proliferation at first day and the socres were 0. Honey group rised on the third and 5th day, and peaked on the 5th day, then declined on the 7th day. This indicated that honey group’peak rised ahead of untreated group and were better than untreated group.③Phenolic acid groups:Peak time rised were later than honey group whose peak rised on the third day and ahead of untreated group whose peak rised on the 7th day. This indicated that collagenous fiber proliferation and blood capillary proliferation in phenolic acid groups were weaker than untreated group but better than honey group.④Different times dose in same phenolic acids:On the first day,2 times group and 1 times group were at the same level which were higher than 0.5 times group in different dose GA groups; GA groups all rised to peak on the 5th day and 2 times were the highest, all declined on the 7th day and 2 times were the lowest, this indicated that 2 times group were the best in anti-collagenous fiber proliferation and anti-blood capillary proliferation. However, PA groups and CA groups all stayed the same changes of increase and decrease, and the score were same at each time point. This showed that collagenous fiber proliferation and blood capillary infiltration didn’t relate with phenolic acid dose.⑤Different kinds of phenolic acids in the same times dose between groups are compared:Scores in all groups were 0. All 2 times group,1 times group or 0.5 times group between the different kinds of phenolic acids were compared. On the first day, scores were 0 in all groups. On third day, the scores in GA groups collagenous fiber proliferation were the highest (P<0.05), this indicated that GA was the best in the three phenolic acids. With the time in varying degrees of increased or decreased, the GA group in three groups of in the lowest position of phenolic acids. This indicated that anti-collagenous fiber proliferation and blood capillary proliferation of GA groups were the strongest in three phenolic acids groups.(2). Result of serum FGF2 content:①Blank group and untreated group:Untreated group were higher than blank group and indicated that serum content of FGF2 rised after wound was established. Untreated group were elvated at each time point, but there were no significance difference between pair-wise comparison, this indicated that in natural wound healing state tissue secrete FGF2 slowly.②Honey group:Honey group were higher than untreated group on the first and third day(p<0.05), rised to the peak, then declined on the 5th and 7th day, and were similar with blank group(p>0.05). This indicated that honey could regulate FGF2 secretion, honey could accelerate FGF2 in early stage, then weaked in middle and late stage, then finished wound healing on 7th day.③Phenolic acids groups:On the first day phenolic acids groups were lower than honey group(p<0.05) but higher than untreated group (p<0.05), this indicated that phenolic acids could accelerate FGF2 secretion when compared with untreatedgroup, but weaker than honey group. On the third day, phenolic acids groups elevated in different degrees, lower than honey group(p<0.05) but higher than untreated group, there were no significance difference between pair-wise comparison in 2 times GA group and different dose of CA groups(p>0.05). This indicated that honey was better than phenolic acids groups. On the 5th day, phenolic acids groups elevated alittle, higher than honey group which was in decline, except no significant difference between 2 times GA group and untreated group(p>0.05), the other groups were higher than honey(p<0.05). This indicated that honey started to weak FGF2 secretion, however phenolic acids groups still accelerated FGF2 seceretion,phenolic acids groups were weaker than honey. Phenolic acids groups were higher than untreated group, except that 2 times CA group vs untreated group(p>0.05)and 1 times group vs untreated group(p>0.05), the other groups vs untreated group(p<0.05), this indicated that phenolic acids groups were better than untreated group in FGF2 secretion. On the 7th day, phenolic acids groups stayed stable with the 5th day, higher than honey group(p<0.05), different dose of GA groups and PA groups were lower than untreated group(p<0.05) but higher than blank group(p<0.05), different dose of CA groups were higher than untreated group(p<0.05) and blank group(p<0.05), this indicated that phenolic acids up-regulate FGF2 seceretion started to weak, GA were better than PA and CA, phenolic acids were weaker than honey group, but better than untreated group.④Different times dose in same phenolic acids:On the first day,2 times phenolic acids groups were higher than 1 times phenolic acids groups which were higher than 0.5 times(p>0.05). On the third day, all groups rised to the peak,2 times phenolic acids groups were higher than 1 times phenolic acids groups which were higher than 0.5 times, GA groups pair-comparison had significant difference(p<0.05), but PA groups and CA groups pair-comparion had no significant difference(p>0.05). Phenolic acids group on the 5th day declined compared with the third day, the 7th day declined compared with the 5th day, at this two time point,2 times phenolic acids groups were lower than 1 times phenolic acids groups which were lower than 0.5 times phnolic groups(p>0.05). This indicated that didn’t relate with phenolic acid dose.⑤Different kinds of phenolic acids in the same times between groups are compared:All2 times group,1 times group or 0.5 times group between the different kinds of phenolic acids were compared on the first day and FGF2 in different dose of GA groups is the highest, PA groups secondly, CA groups the last. However with the time in varying degrees of increased or decreased, the GA group in three groups of in the lowest position of phenolic acids, PA groups secondly, CA groups the last. This indicated that GA was the best, PA secondly, CA the last in wound healing.(3)Result of serum TGFβ1content:①Blank group and untreated group:Untreated group were higher than blank group and indicated that serum content of TGFβ1 rised after wound was established. Untreated group were elevated at each time point, but there were no significance difference between pair-wise comparison, this indicated that in natural wound healing state tissue secrete TGFβ1 slowly.②Honey group:Honey group were higher than blank group(p<0.05) and untreated group on the first and third day(p<0.05), rised to the peak on the third day, then declined on the 5th and 7th day, and on the 7th day were similar with blank group(p>0.05). This indicated that honey could regulate TGFβ1 secretion, honey could accelerate TGFβ1 in early stage, then weaked in middle and late stage, then finished wound healing on the 7th day.③Phenolic acids groups:On the first, third,5th day, phenolic acids groups changed in different degrees, lower than honey group(p<0.05) but higher than untreated group(p<0.05) and blank group(p<0.05), this indicated that phenolic acids could accelerate TGFβ1secretion but were weaker than honey group. On the 7thd day, honey group reached blank group level, different dose of GA groups decreased in different degrees, but all were higher than honey group but lower than untreated group, except that 2 times group vs honey group(p>0.05),1 times group vs honey group(p>0.05),1 times group vs untreated group(p>0.05). Different dose of PA groups were declined in different degrees on the 5th day, higher than honey group(p<0.05), but there were no significant difference between PA groups vs untreated group(p>0.05). Different dose of CA groups stayed with the 5th day, higher than honey group(p<0.05). This indicated that honey group were better than phenolic acids groups, less time to wound healing.4)Different times dose in same phenolic acids:On the first day,2 times phenolic acids groups were higher than 1 times phenolic acids groups which were higher than 0.5 times(p>0.05). On the third day, all groups rised to the peak,2 times phenolic acids groups were higher than 1 times phenolic acids groups which were higher than 0.5 times, GA groups pair-comparison and 2 times PA vs 0.5 times PA had significant difference(p<0.05), but the other groups had no significant difference(p>0.05). Phenolic acids groups on the 5th and 7th day had different changes, at this two time point,2 times phenolic acids groups were lower than 1 times phenolic acids groups which were lower than 0.5 times(p>0.05). This indicated that TGFβ1 didn’t relate with phenolic acid dose.⑤Different kinds of phenolic acids in the same times between groups are compared: both 2 times group,1 times group or 0.5 times group between the different kinds of phenolic acids were compared. On the first day, TGFβ1 in different dose of GA groups were the highest, PA groups secondly, CA groups the last (p<0.05). However, at the same times the number of groups after the third,5th,7th days in varying degrees of increased or decreased, the GA group in three groups of phenolic acids in the lowest position (p<0.05). This indicated that GA was the best, PA secondly, CA the last in regulating TGFβ1.Conclusions:Honey, GA, PA and CA could lead to pro-inflammatory responses, promote collagenous fiber proliferation and blood capillary proliferation, accelerate serum FGF2 and TGFβl expression in early stage when wound is established; lead to anti-inflammatory responses, restrict collagenous fiber proliferation and blood capillary proliferation, weaken serum FGF2 and TGFβ1 expression in middle and late stage. Honey was better than phenolic acids. It couldn’t indicate that there were some certain relations between dose and effect when appeared each item pair-comparison in different times dose in same phenolic acids. Different kinds of phenolic acids in the same times between groups are compared, two times GA group were the best in regulating inflammatory responses, promoting collagenous fiber proliferation and blood capillary proliferation, PA and CA still need to extra study; in regulating the two growth factors, GA was the best, PA the second, CA the last. |
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