The Effects Of Topical Administration Of Exogenous HMGB1 On Ischemic Zone Surrounding Ⅲ° Burns

Objective:To determine its functional effects and optimal dose for wound repair of exogenous HMGB1 on ischemic zone surrounding burns.Methods:Male Sprague-Dawley rats weighting 250–300 g were attained from the Experimental Animal Center and were kept at 12h light-dark cycles with a constant temperature（24±1℃）.All rats were fed a standard diet and fasted for12 h（with free access to water）before experimentation.For HMGB1 treatment,30 rats were randomLy（according to random alphabet）divided into five groups:rats treated with 200,300,400,600 ng of HMGB1 respectively,and a control group treated with PBS（n=6 per group）.This study adopted a previously described rat model of thermal injury.^（2,13）For creating this model,a brass comb（20×25×55 mm,150 g）was used.This comb has four rectangular prongs（10×25 mm）that are divided by three grooves（5mm wide）.The dorsum rats were dehaired with 8%sodium sulfide（dissolved in distilled water）12h before experiment.Before operation,rats were anesthetized with 2%pentobarbital by intraperitoneal injection（diluted in NS）.The brass comb was preheated for 3 minutes in boiling water（100℃）and applied on one side of the dorsum of the rat for 20 seconds to create four full-thickness burns that were divided by three unburned interspaces.The burn sites represent the coagulated zone,the interspaces represent the ischemic zone.The comb was then reheated to 100℃and applied on the other side of the dorsum as described above.Thus,two comb burns were created on either side of the dorsum.For all rats,fluid resuscitation was not given for the burns,but free access to water was allowed.HMGB1 protein（HM-122,HMGbiotech）was subcutaneously injected straight into each interspace,at a concentration of 200,300,400,and 600 ng in 0.1 mL of PBS per group,respectively（n=6 per group）.The control group of 6 burn rats received 0.1 mL of PBS in each interspace by subcutaneous injection.Rats injected with HMGB1 or PBS at time 0（immediately after operation）.Two injections（50μl per injection）were made in each interspace.12 hours after operation,rats were euthanized.Full-thickness biopsies were taken from the interspaces,fixed in formalin for 2 hours,dehydrated,cleared in xylene,embedded in paraffin,and cut into 4-μm sections.For Immunohistochemistry,the following antibodies:purified mouse anti-rat CD31（diluted 1:50 in antibody diluent,monoclonal,NB100-64796;Novus Biologicals,USA）,rabbit anti-rat VEGF（diluted 1:100 in antibody diluent,monoclonal,ab32152;AbCam）,and rabbit anti-rat CD45（diluted 1:100 in antibody diluent,polyclonal,ab10558;AbCam）.VEGF and CD45 proteins extracted from skin tissue were analyzed by Western Blotting.The antibodies and the dilutions were as follows:primary antibody specific for CD45（1:2000 rabbit anti-rat polyclonal,ab10558;AbCam）,VEGF（1:2000,rabbit anti-rat,monoclonal,ab32152;AbCam）.β-actin（Beyotime）was used at a dilution of 1:10000.The RNA level of CD31,CD45,VEGF were analyzed by SYBR green real-time Q-PCR.Total RNA was extracted from continuously liquid-nitrogen frozen,pulverized skin tissues using TRIzol reagent（TIANGEN）following the manufacturer’s instructions.The cDNA was amplified and detected after determining the optimal cycling conditions.Results:Immunohistochemistry stain（200×）for the tissues revealed that the expression of CD31 was significantly higher in 200 ng group compared to 300,400,600 ng and control group（P<0.01）.The expression of CD31 decreased when using a greater dose of HMGB1 on unit ischemic area（P<0.01）,but 600ng group was no difference to the PBS-treated group（P>0.05）.CD31-positive vessels were counted to determine the amount of microvessels,including neovascularization.The microvessel density（MVD）in ischemic zone of 200 ng group was higher,compared to 300,400,600 ng and the control group（P<0.05）.With an increasing does of HMGB1,the MVD decreased instead,but400,600 ng and control group were no difference to each other（P>0.05）.The immunohistochemistry stain（100×）showed that the VEGF expression was significantly increased in all HMGB1-treated groups compared to control group（P<0.05）.In 200 ng group,the expression of VEGF was higher than that of 300,400 and 600 ng group（P<0.01）,while the 400,600 ng group were no difference to each other（P>0.05）.The westernblot analyses demonstrated that the VEGF protein level was significantly increased in 200 ng group when compared with 300,400,600 ng and control group（P<0.01）,but the results of400,600 ng and control group were not statistically significant（P>0.05）.The real-time PCR showed that the mRNA expression of VEGF was significantly elevated in 200 ng group（P<0.01）.The mRNA expression of VEGF in 300,400 ng group were higher than 600 ng and control group（P<0.01）,while there was no difference in 300 and 400 ng group（P>0.05）,the 600 ng group was also no difference to the control group（P>0.05）.The immunohistochemistry stain（100×）showed that the expression of CD45 was upregulated in all HMGB1-treated groups compared to PBS treated group（P<0.01）.The expression of CD45 in 300 and 600 ng group were higher than that in 200 and400 ng group（P<0.01）,and it was even higher in 300 ng group compared with600 ng group（P<0.01）,while there was no difference in 200 and 400 ng group（P>0.05）.The westernblot data showed that the protein level of CD45 in300,600 ng group was higher than that in 200 ng and control group（P<0.05）,but in 300 ng group it was not statistically significant to the 400,600 ng group（P>0.05）.The real-time PCR data showed that the expression of CD45was significantly elevated in 300,400 ng group,compared to 200 and 600 ng group（P<0.01）.The control group was higher than 200 and 600 ng group（P<0.05）,but was no difference to 400 ng group（P>0.05）.Conclusion:Topical administration of exogenous HMGB1 is beneficial in early phase of ischemic zone progression of thermal injury on rats.By promoting angiogenesis,this beneficial effect reaches a peak when subcutaneously injecting 200 ng/0.1mL of HMGB1.Exogenous HMGB1promotes ischemia-induced angiogenesis in the ischemic zone possibly via a VEGF-dependent mechanism.The therapeutic effect of HMGB1 decreases with the increase in its dose,it is likely that the increasing dose of HMGB1 reinforce the inflammatory response,by which the angiogenesis function is impaired.