| Objective1.The experiment observed the effect of Hatageqi-7 therapy of Mongolian medicine on pressure ulcers with ischemia-reperfusion injury in rats in different aspects to clarify the mechanism of treating pressure ulcer2.To establish the rat model of ischemia-reperfusion injury of pressure sores and observe the influence.of Hatageqi-7 powder on wound healing,To analyze the mechanism of wound healing and provide the experimental basis for clinical application and experimental data.Methods1.Animal screening:Buy 80 healthy male SD rats of clean grade,weight 280 ±20g.According to random number table method,the rats were randomly divided into four groups:sham operation control group,model group,Hataqeqi-7 group,Yunnan Baiyao group,with 20 rats in each group.2.make rat model on I/RI of pressure ulcer:General anesthesia of rats using 10%Chloral hydrate with 300mg/kg,cleaning and disinfecting after hair removal in hip of rats.Perpendicular to the spine to make a 2.5cm incision in the right hip,blunt separation and implant 25 mm x 1.5 mm diameter circular magnet of hip skin deep fascia,sewn up last 4-0 full thickness incision.24h after surgery,a size of 25mm X 2.5mm of the permanent magnet,and began to skin the ischemia-reperfusion injury cycle.Absorbed oin the hip of a piece of magnet in rats implanted area for 2 hours,then remove skin blood flow and reperfusion for 30 minutes in the area,this is a cycle.Be repeated 5 cycles a day for 5 days.To sham operation control group,only do incision on the same position of the hip in rats.Depth of the incision reaches the deep fascia.Finally,skin darkening,harden,not the bleeding as a criterion for judging ulcers.3.Rats grouping and therapies:Selected 16 rats of each group from Model group,Hatageqi-7group and Yunnan Baiyao group with the phase III pressure ulcer model in rats.Selected 16 rats from sham operation control group according to random number table method.Each group divided 7 days and 14days of two time points,each time group has 8 rats.The treating groups give corresponding drugs,one time/day,with sterile gauze to cover the wound and bandage fixed wrap.Sham operation control group and model group does not give any way of treatment.4.Materials drawing and index detection:? generally observation:to observe the wound of rats,such as wound color of rats,exudate and calculate rat wound area.?After anesthesia of rats in the each period time,to collect 5ml of abdominal blood and in a be without anticoagulant,with 3000r/min,4? centrifuge for 10 min,and take serum.According to the kit.to detect serum SOD,MDA,MPO,and NO content,and TNF-?,ICAM-1 levels through double antibody sandwich enzyme-linked immunosorbent determination.? cutting the left side of the full skin and muscular tissue in pressure ulcer.4%paraformaldehyde fixed,dehydrated,conventional paraffin embedding,HE staining for morphological observation and the expression of HIF-1?and VEGF was detected by Immunohistochemistry method.? cutting the right side of the full skin and muscular tissue in pressure ulcer,and take into frozen storage tube,save to-80? refrigerator.The amount of HIF-la and VEGF mRNA was detected by Reverse transcription polymerase chain reaction.The expression of HIF-la and VEGF were detected by Western blot method.5.Statistical treatment:using the SPSS 21.0 software for statistical treatment.And the data is showed mean ± standard deviation.Comparison between groups using F test.LSD test comparison between the two groups.P<0.05 has statistical significance.Results1.General observation of wound healingThe wound exudate of Hatageqi-7 group was more.Granulation tissue growth was rapid,bright color reddish.To the naked eye view,the wound began to heal from 3 days and heal in 14 days.The wound exudate of YunnanBaiyao group was moist.Granulation tissue growth was rapid,pink color.To the naked eye view,the wound began to heal from 5 days and heal in 14 days.The wound exudate of model group with physiological saline moist,to avoid tear at the bleeding,wound healing slowly,the wound began to heal from 7 days,until the 14th day still healing rate is not high.2.The effect of Hatageqi-7 for wound healing of pressure ulcers with ischemia reperfusion injury in ratsThe residual pressure ulcer area between different time points is as follows:for 3 days,there is no significant difference between groups of rats(P>0.05).But for 7 and 14 days,compared with the model group,the residual pressure ulcer area on groups of Hatagqi-7 and YunnanBaiyao were significantly reduced(P<0.01).Each time point.there was no significant difference between Hatagqi-7 group and YunnanBaiyao group(P>0.05).It means Hatageqi-7 group and Yunnan Baiyao group have better repair effect on pressure ulcers with Ischemia-reperfusion injury in Rats.3.The influence of pathological changes on pressure ulcers with Ischemia-reperfusion injury in ratsSham operation control rats skin belongs to stratified squamous epithelium,epithelial tissue structure is clear,muscle fibers are arranged closely and orderly,not seen obvious inflammatory cells infiltration.model group rats skin surface is not complete,stratified squamous epithelium is thinner than the normal group,the structure is not clear,part of epithelial structures appear rupture,muscle fibers are edema,interstitial hyperemia,intradermal see more inflammatory cells infiltration.Epithelium structure of Hatageqi-7 and YunnanBaiyao group is clear,can relieve muscle fiber edema,and no congestion between organizations,a small amount of inflammatory cells infiltration can be seen in the dermis.It showed effect of Hatageqi-7 on reducing ? period of pressure ulcer ' s inflammatory infiltration were more apparent.4.The effect of serum SOD?MDA?NO?MPO?TNF-? and ICAM-1 on pressure ulcers with ischemia-reperfusion injury in rats4.1 Effect of SODEach time point,compared with Sham operation control group,serum SOD content in model group was significantly decreased(P<0.01).Compared with model group,serum SOD contents of Hatageqi-7 powder and YunnaBaiyao groups were significantly increased(P<0.01).4.2 Effect of MDAEach time point,compared with Sham operation control group,serum MDA content in model group was significantly increased(P<0.01).Compared with model group,serum MDA contents of Hatageqi-7 powder and YunnaBaiyao groups were significantly decreased(P<0.01).There was no statistically significant difference between Hatageqi-7 group and YunnanBaiyao group(P>0.05).4.3 Effect of NO and MPOEach time point,compared with Sham operation control group,serum NO and MPO contents in model group was significantly increased(P<0.01).Compared with model group,serum NO and MPO contents of Hatageqi-7 and YunnaBaiyao groups were significantly decreased(P<0.05).Compared with YunanBaiyao group,7 days the content of serum MPO of Hatageqi-7 group was significantly decreased(P<0.05).4.4 Effect of TNF-? and I CAM-1Each time point,compared with Sham operation control group,serum TNF-?and ICAM-1 contents of model group was significantly increased(P<0.01).Compared with model group,serum TNF-a and ICAM-1 contents of Hatageqi-7 and YunnaBaiyao groups were significantly decreased(P<0.01).Compared with YunanBaiyao group,serum TNF-a and ICAM-1 contents of Hatageqi-7 group was significantly decreased(P<0.05).5.Effect of HIF-la and VEGF of wound tissue on pressure ulcers with ischemia-reperfusion injury in rats5.1 RT-PCR results:Each time point,compared with sham operation control group,HIF-la and VEGF mRNA expression of each groups were significantly increased(P<0.01).Compared with model group,HIF-la and VEGFmRNA expression of Hatageqi-7 and YunnaBaiyao groups were significantly increased(P<0.05).compared with Hatageqi-7 group,VEGF mRNA expression of YunnaBaiyao groups was significantly increased(P<0.05).5.2 Western blot results:Each time point,compared with sham operation control group,HIF-l? protein expression level of each groups were significantly increased(P<0.01.Compared with model group,HIF-la and VEGF protein expression level of Hatageqi-7 and YunnaBaiyao groups were significantly increased(P<0.05).There was no statistically significant difference between Hatageqi-7 group and YunnanBaiyao group(P>0.05)?Each time point,compared with sham operation control group,VEGF protein expression level of each groups were increased,VEGF protein expression level of Hatageqi-7 and YunnaBaiyao groups were significantly increased(P<0.01).Compared with model group,HIF-la and VEGF protein expression level of Hatageqi-7 and YunnaBaiyao groups were significantly increased(P<0.01).After 14 days later,compared with YunanBaiyao group,VEGF protein expression level of Hatageqi-7 group was significantly increased(P<0.05).5.3 IHC results:Each time point,compared with sham operation control group,HIF-la and VEGF protein expression level of each groups were significantly increased(P<0.05).Compared with model group,HIF-la and VEGF protein expression level of Hatageqi-7 and YunnaBaiyao groups were significantly increased(P<0.05).There was no statistically significant difference between Hatageqi-7 group and YunnanBaiyao group(P>0.05).Conclusion1.Hatageqi-7 could significantly reduce the wound area on pressure ulcers with ischemia-reperfusion injury.At the same time,the pathological results showed that it could reduce inflammation cells infiltration of the wound tissue.This showed Hatageqi-7 had a repair effect on wound healing,and effectively promoted the healing of wounds.2.Hatageqi-7 could improve SOD activities,reduce the content of serum MDA and NO,reduce lipid peroxidation,improve antioxidant enzyme activities,and remove free radicals,so to promote wound healing.3.Hatageqi-7 could the levels of TNF-a.MPO and ICAM-1 of rats serum on ulcers with ischemia-reperfusion injury.The mechanism may be related to the inhibition of inflammation caused by pressure ulcer,and promote the wound healing.4.Hatageqi-7 could improve expression of HIF-la and its downstream factor VEGF protein in the wounded skin tissue,reduce the ischemia-reperfusion injury and improve the local blood circulation and promote the proliferation of blood vessels and fibrous tissue,and thus promoted the repair of the wound. |
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