Bacille Calmette-Guerin(BCG) Or BCG-PSN By Inhalation Affected β-defensins And Toll-like Receptor Gene Expression Of Respiratory Tract

The scholars pointed out that drug-resistance bacteria have becomemain pathogenic microorganism of severely threatening health of mankindon the 21st century. Because the number of sensitive antibiotics treatinginfectious disease will gradually decreased, no sensitive antibiotics can besometimes used ,for solving the difficult problem of the whole world, thescholars at home and abroad transfer research emphasis on enhancing naturalimmunity of body to assist anti-infection and decreasing the number ofantibiotics use. Nonspecific immunity is the first barrier of organism againstinfectious diseases. TLR was recently discoveried a human homologue of thedrosophila Toll protein.As recognization pathogen and fast reaction protein,Toll-like receptors played an important role in innate immunity.In 1997,Medzhitov and colleagues were the first to characterize a human TLR(latercalled TLR-4).To date,ten TLR family members(TLR1-10) have beenidentified in the human genome. TLR is a type I transmembrane protein,withan ectodomain consisting of leucine-rich repeats(LRRs) and one or twocysteine-rich regions. The intracellular domain of Toll-releated receptorscontains a Toll/ IL-1 receptor(TIR) domain,based on homology of the regionwith a similar intracellular domain of the IL-1 receptor(IL-1R),known asTLR(Toll/IL-1R) structural domain. Toll-like receptors provides us with thelong sought transmembrane molecules linking the extracellularcompartment ,where contact with and recognition of microbial pathogensoccurs,and the intracellular compartment,where signaling cascades leading tocellular responses are initiated. TLR can recognize cell wall constituent ofpathogenic microorganism(as LPS , PGN),and initiate cell personalanti-infection immunity. TLRs are mainly expressed in lymphoid tissues suchas macrophage, dendritic cell,neutrophils. Different TLRs are also expressedin non-lymphoid tissues .It has recently been shown that TLRs1～6 areexpressed in the epithelial cells of human respiratory tract. It has recently beenshown that TLR-2 possess multi-ligands, such as peptidoglycan andLipoteichoic acid from Gram-positive bacterial cell wall, glucolipid ,lipoprotein , lipopeptide from mycobacterial species and sprirochete,polysaccharide from fungi. TLR-4 was shown mainly to recognizelipopolysaccharide from Gram-negative cell wall.Thus ligands for TLR-2 andTLR-4 cover most microorganisms that are pathogenic to human body in thenatural world.Therefore, TLR-2 and TLR-4 are the most important receptors inhuman TLR family members.Respiratory tract epithelia secreting antibacterial peptides that play animportant role in the natural defences of most living organisms againstmicrobial infection. Especially β-defensins, natural chemistry barrier are oneof important advances of antimicrobial peptides in innate immunity in thelate 10 years. β-defensins have a wide range of antimicrobial acitivity in vitroencompassing Gram-negative and Gram-positive bacteria, fungi,and viruses.β-defensins are proposed to participated in the early host defense responseagainst microorganisms in vivo. Except for naturally resistant bacteria:Burkholderia cepacia and Serratia marcescens, β-defensins do not induceacquired drug-resistance on other pathogenic microorganisms. Therefore,bacterial resistance occurs in antibiotics that has become a severe problem ofthe whole world today , energetic elevation innate immunity of organism stillto remain emphasis after research, the domestic and abroad scholars paydouble attention to exploratory developments of foundation and preclinicalphase of β-defensins.In addition, β-defensins possess immunoregulation function. HBD-1mRNA,hBD-2mRNA,hBD-3 mRNA are all expressed in the epithelial cells.HBD-1 mRNA is mainly expressed in the renal epithelial cells;hBD-2mRNA,hBD-3 mRNA are mainly expressed in the inflammatory skin;hBD-1 mRNA,hBD-2mRNA,hBD-3 mRNA are all expressed in the respiratory tract. Cellculture in vitro demonstrated hBD-2mRNA,hBD-3 mRNA were upregulatedby IL-1β,TNF-α,bacteria, fungi and LPS. How to produce β-defensins inepithelial cells of air passage? As recognition pathogens and fast reactionproteins, TLRs play an important role. Recently, Wang et al finding thatup-regulation hBD-2 of airway epithelia was finished through pathway oftoll-like receptor 2 mediated NF-κB/IκB. At present, biological function ofβ-defensins was mostly studied in vitro. If some drugs can enhance geneexpressions of β-defensins in vivo, accordingly anti-infection therapy ,drug-resistance bacteria constitute serious threaten of mankind health on the21st century,further researchs of β-defensins have significant theory andpractical importance.In a word,medical circle gradually recognizes polluted air and organicdust containing endotoxins are important causes of environmental andoccupational respiratory diseases ,lipopolysaccharide is the principalconstituent of endotoxin, derived from Gram-negative bacteria cell wall. BCGor BCG-PSN is a diphasic immunomodulator. In addition, a number ofresearchs showed respiratory tract epithelia expressing TLR2 , TLR4mRNA,which played important biological functions in innate immunity andadaptive immunity. Otherwise, although defensins possess broad spectrumantimicrobial activity ,and contact pathogenic bacteria again and againwithout inducing acquired drug-resistance on other pathogenicmicroorganisms,all that is paid close attention , which drugs can inducesafely and effectively increasing endogenous gene expression of β-defensinsto treat infectious diseases in vivo,that is only an area of beginning .If geneexpressions of TLR2,TLR4 and β-defensins are further clarified in normal,inflammatory,immunoregulatory conditions, it may provide effective andnew method to treat infectious diseases.To confirm if BCG or BCG-PSN by inhalation reinforced rat withbronchopneumonia β-defensins and Toll-like receptor gene expression inlung,the rats were intratracheally instilled with LPS, 1 mg/kg and raised for3 d. Food-intake,drinking water,respiratory rate of the rats were closelyobserved in the meantime. On fourth day: rats were killed. The anticoagulatedblood of peripheral blood,bronchoalveolar lavage fluid were used to leucocytecount and differential count. Hereafter a portion of right lung was made formicroscopic pathological specimen. The rat model with bronchopneumoniawas confirmed to make successfully.Subsequently,every rat inhaled 3.3ampoule of BCG or BCG-PSN or 3.3ml of normal saline each time. Theinhalation was done at a time every other day. Food-intake,drinkingwater,respiratory rate of the rats were closely observed in the meantime.The rats in every subgroup were killed respectively on the second day ofthe inhalation of 3 times,5 times,7 times. Gene expression of β-defensin-1,β-defensin-2, TLR2,TLR4 in bronchopneumonic rat, in normal,inflammatory conditions and before and after BCG or BCG-PSN by inhalationin lung in vivo were studied by RT-PCR analysis. If gene expression ofβ-defensin-1,β-defensin-2, TLR2,TLR4 in bronchopneumonic rat inducedLPS in vivo was affected by BCG inhalation. In order to discuss if it ispracticable for increasing endogenous gene expressions of β-defensins andToll-like receptors in vivo by BCG inhalation.Thereby,if it may providetheoretical foundation to treat infectious diseases by increasingendogenous gene expression of β-defensins and Toll-like receptors.The following results were obtained by contrastive studies ofnormal rats and pneumonic rats, BCG-PSN group and normal salinegroup ,BCG group and normal saline group, BCG group and BCG-PSNgroup;contrastive studies of before and after BCG or BCG-PSN ornormal saline by inhalation and normal rats and pneumonic rats:1. The models of rat with bronchopneumonia were manufactured byintratracheal injections of LPS one time. The models of rat withbronchopneumonia were determined doing well by clinical manifestation andwhite-cell count and differential count of peripheral blood andbronchoalveolar lavage fluid ,the changes of pathology in bronchi and lung .2. TLR2 mRNA expression in bronchopneumonic rat by inhalation ofBCG-PSN: compared with normal control, at inhalation of 5 times, there wassignificant difference(p<0.05),at inhalation of 7 times ,there was moresignificant difference(p<0.01);compared with pneumonia model group orafter inhalation of normal saline in bronchopneumonic rats, at inhalation of 7times, there was significant difference (p<0.05), at inhalation of 3 or 5 times,there was no significant difference (p>0.05).TLR2 mRNA expression inbronchopneumonic rat by inhalation of BCG: compared with normalcontrol,pneumonia model and normal saline group, at inhalation of 3 times,there was significant difference(p<0.05), at inhalation of 5 or 7 times, therewas no significant difference (p>0.05).All results showed that bothBCG-PSN and BCG can enhance TLR2 mRNA expression,but BCG-PSNgroup compared with BCG group,the former became effective slowly ,moreefficiently and longer-acting and in a time-dependent manner.BCG inhalationcan slightly increase TLR4 mRNA expression in lung in bronchopneumonicrats, at inhalation of 5 times, TLR4 mRNA expression was the highest, therewas no obvious difference in all groups(p>0.05). BCG-PSN did notinfluence TLR4 mRNA expression in lung in bronchopneumonic rats.3. β-defensin 1 mRNA was expressed in normal rats andbronchopneumonic rats,at levels that did not differ significantly(p>0.05);BCG or BCG-PSN by inhalation can not enhance β-defensin 1 mRNAexpression of rat with bronchopneumonia in lung;BCG-PSN groupcompared with normal saline group, BCG group compared with normalsaline group, BCG-PSN group compared with BCG group: there wasno obvious difference in RBD-1 mRNA expression of lowerrespiratory tract(p>0.05).β-defensin 2 mRNA expression in pneumoniamodel group was higher compared with normal control group, there wassignificantly difference between two group(sp<0.05). BCG or BCG-PSN byinhalation can enhance β-defensin 2 mRNA expression of rat withbronchopneumonia in lung;β-defensin 2 mRNA expression inBCG-PSN group or BCG group was significantly higher compared withnormal saline group(p<0.05),and RBD-2 mRNA expression reachedpeak value at inhalation of 5 times;there was no significant difference inRBD-2 mRNA expression in BCG-PSN group compared with BCGgroup, at inhalation of different times in BCG-PSN group or BCG group(p>0.05).4. Food-intake,drinking water,respiratory rate of bronchopneumonicrat in BCG-PSN group or BCG groups recovered before that ofbronchopneumonic rat in normal saline groups(p<0.01). It indicated BCGor BCG-PSN by inhalation played the role of promoting disappearing andabsorption of bronchial and pulmonary inflammation, and promotingdisappearing of clinical symptoms of rats with bronchopneumonia.Conclusion1. BCG or BCG-PSN by inhalation can enhance TLR2 mRNA expression,BCG inhalation can slightly increase TLR4 mRNA expression in lung inbronchopneumonic rats, BCG-PSN did not influence TLR4 mRNA expressionin lung in bronchopneumonic rats. These results explained general tendency ofBCG or BCG-PSN by inhalation affecting rat with bronchopneumonia TLR4and TLR2 gene expression in lung was uniformity, but there was differencein effect. These results suggest that BCG or BCG-PSN can stimulate TLR2mRNA expression in lung of bronchopneumonic rats ,assist stimulatingnonspecific immunity ,thereby help to anti-infection and reduce the number ofantibiotics use . In addition , BCG-PSN group compared with BCG group,theformer became effective slowly,more efficiently and longer-acting and in atime-dependent manner.These results illustrated different components ofmycobacterium tuberculosis educed different effects in adjusting TLR2 mRNAexpression.2. Rat with bronchopneumonia by inhalation of BCG or BCG-PSN,β-defensin 1 mRNA expression in lung can not be enhanced,butβ-defensin 2mRNA expression can be enhanced. It demonstrated that β-defensin 1 mayplay a constitutive role in respiratory defense;whereas RBD-2 may beinduced in response to local infection or inflammation, namely RBD-2 mRNAexpression at low levels under normal condition;stimulation with infection orimmunoregulator RBD-2 mRNA expression enhanced.These results suggestthat BCG or BCG-PSN can stimulate lung of rat with bronchopneumonia invivo, kill effectively foreign pathogenic bacteria in time by secretingendogenousβ-defensin 2, educe natural barrier function in the earlyInflammatory reaction.3. BCG or BCG-PSN by inhalation played the role of promotingdisappearing of clinical symptoms of rats with bronchopneumonia.Its causewas considered BCG or BCG-PSN enhanced TLR2 and RBD-2 mRNAexpression, triggered cascade reaction of cell signal,activated NF-κBtranscription factors binding to the corresponding region of the respectivetrachea antibacterial peptide (TAP) gene .RBD-2 mRNA expression in lungwas induced by inhalation of BCG or BCG-PSN to undertake anti-infectivetherapy. According to the latest view of upper ,lower respiratory tractdependability,our results also have important reference values on upperrespiratory tract inflammatory diseases.