Study On The Activity Of High Molecular Weight Hyaluronic Acid On LPS-induced Acute Lung Injury And The Mechanism

Objective: Acute lung injury is a clinical critical illness,however,there is no specific medicine for teatment.Hyaluronic acid is an endogenous substance,in addition to a strong moisturizing effect,hyaluronic acid also contributes to antitumor and immune regulation.But there is less report about the protection of hyaluronic acid on acute lung injury.It is important to study whether HA has therapeutic effect on acute lung injury and study about the anti-inflammatory mechanisms.Methods:(1)Cell experiments: q PCR and ELISA were used to detect the mRNA and protein expression of IL-6,IL-8,TNF-α and IL-1β in 3,6,12,24 h after LPS stimulation with different concentration(1,10,50,100 μg / mL)in A549 cell to explore the optimal concentration and stimulation time of LPS.Insure the acute lung injury model was established successfully.Specific high molecular weight hyaluronic acid was used to cure the A549 cell acute lung injury model at the concenttation of 0.3,0.5,0.7,1 mg/mL.The mRNA and protein expression of IL-6,IL-8,TNF-α and IL-1β were detected by qPCR and ELISA.The dose-effect relationship of hyaluronic acid was explored.(2)Animal experiments: SD rats were randomly divided into control rats;LPS(5mg/kg)rats;LPS with HA(3.5mg/mL)rats.The right lung was fixed,sliced and stained by HE for pathological observation.SD rats were randomly divided into control rats;LPS rats;LPS with HA(1 mg/mL)rats;LPS with HA(2 mg/mL)rats;LPS with HA(3.5 mg/mL)rats.The bronchoalveolar lavage fluid of left lung was collected for counting neutrophils and monocytes after centrifugal.We investigate the effect of HA on the mortality of LPS-induced acute lung injury in rats.(3)The mechanism of Hyaluronic acid’s treatment effect on acute lung injury was investigated on the molecular level.The mRNA expression of TLR4 was detected by RT-PCR and qPCR.Western Blot was used to detect the expression of protein IκB-α in cytoplasmic and protein NF-κB p65 between cytoplasm and nucleus.Results:(1)The A549 acute lung injury model was successfully established after LPS stimulation at the concentration of 1μg / mL;q PCR and ELISA showed that hyaluronic acid was effective on the treatment of acute lung injury,and the effect increased significantly in the effective concentration range of 0.3~0.7 mg/mL.(2)Compared with the acute lung injury model group induced by LPS,HA treatment group was clear and complete,the infiltration of neutrophils and monocytes decreased,the amount of neutrophils and monocytes decreased,and the data showed HA dose-dependent,indicating that HA treatment reduced the inflammation caused by LPS induction.HA treatment group decreased mortality and showed HA dose-dependent.(3)RT-PCR and qPCR showed that HA treatment inhibited the mRNA expression of TLR4,and the results of Western Blot showed that HA treatment promoted the expression of IκB-α protein and inhibited NF-κB p65 protein transfering from cytoplasm to nucleus.Conclusion: The results of experiments in vitro and in vivo showed that high molecular weight hyaluronic acid had therapeutic effect on LPS-induced acute lung injury and showed dose-dependent.Specific high molecular weight HA may interfere with LPS-induced A549 inflammatory response through the NF-κB signaling pathway.