Study On The Expression Characteristics And Mechanism Of Autophagy Protein In Tuberculous Wound

Objective:1.By studying the expression rules and characteristics of autophagy related proteins Beclin1 and LC3B in tuberculous wound,the role of autophagy mechanism in delayed healing and high recurrence rate of tuberculous wound was preliminarily revealed,and the abnormal expression of autophagy related proteins Beclin1 and LC3B may be one of the key mechanisms in the pathogenesis of tuberculous wound.2.By studying the injection of freund’s complete adjuvant(FCA)can induce autophagy tuberculous wound tissue and its influence on tissue pathological changes,to explore the FCA autophagy protein expression in tuberculous wound,tries to reveal autophagy ties with tuberculous wound pathogenesis mechanism and for further research on tuberculosis wound pathogenic mechanism provides a new train of thought,seeking to promote new ways of the treatment of tuberculous wound healing.Methods:1.Beclin1 and LC3B expression levels in tuberculous wound tissues,chronic wound tissues and normal skin tissues were detected by immunohistochemistry.2.Choose 12 male SD rats,each subcutaneous injection of 0.2 mL sensitization,FCA sensitization after 3 weeks per subcutaneous injection of BCG only 0.2 mL in infection(1x10~7/mL,bacteria liquid concentration),afterinfection,respectively 8 d,15 d,32d 43,d random executed three focal infection were taken and injection of FCA in organization,HE dyeing method to observe the local pathological changes,immunohistochemical method in the analysis of local tissue Beclin1,LC3B protein expression.3.Twenty male SD rats were selected,each of which was sensitized by subcutaneous injection of 0.2ml of FCA,and each of which was sensitized by subcutaneous injection of 0.2ml of BCG after 1 week of sensitization(concentration of bacterial solution:1×10~7/mL).After infection,the rats were divided into BCG group(n=5rats,blank control group).BCG+Rapamycin(RAPA)group(n=6,autophagy enhanced control group):after BCG infection,intraperitoneal injection of RAPA was given once every other day at 3mg/kg.BCG+3-methyl adenine(3MA)group(n=6 individuals,autophagy inhibition control group):BCG infection was followed by intraperitoneal injection of 3MA 3mg/kg every other day.BCG+starvation group(n=3,autophagy positive control group):after BCG infection,48h after infection,starvation caused body weight loss,followed by normal feeding.On the 7th day of infection,all the rats were killed,and the infected foci and FCA tissues were injected.In addition,three untreated rats were sampled as normal group.HE staining was used to observe local pathological changes,and immunohistochemistry was used to analyze the expressions of Beclin1 and LC3B proteins in local tissues.Results:1.Beclin1 and LC3B protein contents in human tuberculous wound tissues were different from those in normal skin and non-tuberculous chronic wound tissues.Abscess was observed in the tuberculous wound tissues of rats at the site of BCG injection.With the extension of infection time,Beclin1,LC3BmRNA content gradually increased on the8day and 15day of infection,reached the highest value on the 32day,and decreased on the 43day.Beclin1,LC3B protein expression and LC3B/A ratio increased gradually with the extension of infection time on 8day and 15day(Beclin1,P<0.01,),decreased on32day and increased again on 43day.2.Beclin1 and LC3B protein expression in BCG group was higher than that in normal group(P<0.05).Rapamycin can reduce mRNA transcription level of LC3B protein in infected foci,and 3MA can increase Beclin1 mRNA transcription level in infected foci.LC3B expression levels of BCG group,BCG+RAPA group and BCG+3MA group were successively decreased,and Beclin1 protein expression level of BCG+3MA group was higher than that of BCG group and BCG+RAPA group.No local inflammatory reaction was observed at the injection of FCA,and inflammatory cell infiltration was observed by HE staining.With the extension of infection time,LC3B gradually increased and then decreased,gradually increased on8day and 15day,peaked at 32day,and decreased on 43day,with no significant change in Beclin1.The intervention factors could affect Beclin1 and LC3B expression in the tissues injected with FCA.Beclin1 expression was significantly increased in BCG group,BCG+RAPA group and BCG+3MA group,while LC3B expression was significantly increased in BCG group,with statistically significant difference(P<0.05).Conclusion:1.Beclin1 and LC3B protein expression levels in the tuberculous wound gradually increased with the extension of infection time and then fell back down on the 32 day and then increased again,which may be one of the reasons why the tuberculous wound repeated attacks and delayed healing.2.mproving the body’s ability to clear tuberculous bacteria by intervening autophagy may be a potential method to treat tuberculous wound.3.FCA can be used as an inducer to induce autophagy in tuberculous wound,which is beneficial to play the pathogenic role of inhibiting tuberculosis bacteria by enhancing autophagy,and may be a potential therapeutic approach for tuberculous wound.