Molecular Mechanism Of Low Molecular Mass Hyaluronan-induced Pulmonary Inflammation In Mice

Objective:Low molecule mass hyaluronan（LMMHA）,a result of rapid turnover and degradation of hyaluronan.Although the accumulating data that support the LMMHA has been implicated in pulmonary inflammatory diseases,the signaling pathway of LMMHA（200 kDa）that initiates the inflammatory response in lung has not been completely investigated.Hence we investigated the role of the PI3K and its downstream signal pathway in LMMHA（200 kDa）-induced lung inflammatory responses.These studies will surely lend a new perspective to our understanding of molecular mechanisms involved in pulmonary diseases.Methods:Here in vivo system by intratracheal administration of LMMHA（200kDa,65 mg/kg）was used to mimic the stressed environment where LMMHAs are amply produced.The bronchoalveolar lavage fluid（BALF）was collected and the total cell and neutrophils were counted.Neutrophils were detected with a histochemical stain for naphthol-ASD-chloroacetate esterase.MPO activity was analysed,and the levels of IL-6,KC and pro-MMP9 in the BALF were measured with ELISA kits to further demonstrate whether LMMHA induced pulmonary inflammation.In the molecular mechanism study of LMMHA-induced pulmonary inflammation,Akt1knockout mice or WT mice were pretreated with PI3K specific blockers（wortmannin or LY294002）or PKCδspecific inhibitor（rottlerin）for 30 minutes before intratracheally LMMHA administration.Mice were sacrificed at 6 h for functional analysis.The phosphorylation levels of Akt1,ERK and P38,and the expression of Mcl-1 and PKCδprotein were detected by Western Blot.The relationship between LMMHA-induced pulmonary inflammation and PI3K/Akt1/PKCδsignal was investigated by flow cytometry,real-time PCR and caspase-3 activity.Results:（1）The intratracheal administration of LMMHA（200 kDa）resulted in a remarkable increase in total cell and neutrophil counts,induced the expression of inflammatory mediators IL-6,KC and pro-MMP9 was increased significantly in BALF of mice;The results demonstrate a substantial increase in MPO activity in LMMHA-treated mice 6 h after LMMHA administration;The staining of lung tissue consistently indicates that the neutrophils infiltrate the lung in response to LMMHA.（2）The pharmacological inhibition of PI3K or genetic deletion of Akt1 enhances neutrophil apoptosis,attenuates neutrophil influx into the lungs of mice and diminishes the expression of pro-inflammatory factors after LMMHA administration.（3）The apoptosis of lung neutrophils was significantly increased in PI3K inhibits or Akt1^-/-mice,which proves to be largely mediated by the anti-apoptotic protein Mcl-1.（4）Further study using PKC-specific inhibitor,rottlerin,revealed that abrogation of PKCδactivity was remarkably decreased LMMHA-induced neutrophil counts and the expression of pro-inflammatory factors in BALF.Conclusions:（1）These results demonstrate that LMMHA（200 kDa）serves as a critical signal to activate and attract neutrophils;The intratracheal administration of LMMHA causes pulmonary neutrophil inflammation and results in neutrophils infiltration into the lungs of the mice.（2）PI3K/Akt1 signaling is critical in LMMHA-induced neutrophilic inflammation.（3）LMMHA-challenged PI3K/Akt1signaling through the up-regulation of the expression of the anti-apoptotic protein Mcl-1 delays lung neutrophil apoptosis.（4）LMMHA induces activation of PKCδsignal to promote neutrophil infiltration,aggregation and release of inflammatory mediators,which enhances pulmonary inflammatory response.The studies demonstrate for the first time that pharmacological inhibition of PI3K or PKCδ,or genetic deletion of Akt1 alleviates LMMHA-induced pulmonary inflammatory response.More importantly,the PI3K/Akt1 pathway participates in LMMHA-induced inflammatory responses,which mainly up-regulates the expression of Mcl-1 to inhibit neutrophil apoptosis.Hence,inhibition of the PI3K/Akt1 pathway exerts a protective role in LMMHA-induced lung inflammation.These new findings reveal a novel molecular mechanism underlying sterile inflammation and provides a new potential target for the treatment of pulmonary disease.