| Objective: Acute lung injury(ALI)is an emergency and severe disease with high fatality.As a means of treatment,mechanical ventilation(MV)often causes ventilator-induced lung injury(VILI)and ventilator associated pneumonia(VAP),which lead to secondary injury and ALI aggravation.High molecular weight hyaluronic acid(HMWHA,1600 k Da)inhibits inflammation in the lungs and is expected to be used as an ALI treatment.Due to the high price of HMWHA(1600k Da),one of the purposes of this experiment is to obtain the HMWHA(1600 k Da)with narrow distribution by separating and purifying the low-cost wide-distribution HMWHA.The other one is to verify the role of HMWHA on in vivo and in vitro ALI models built by MV synergies with low molecular weight hyaluronic acid(LMWHA),further ensure the feasibility of this separation and purification method.Methods:(1)Separation and purification methods: using semi-preparative liquid chromatography,high-pressure homogenization and gel filtration chromatography to separate HMWHA with a molecular weight of about 1600 k Da,based on molecular weight size,molecular weight distribution and method stability Etc.as indicators to determine the best separation method and condition parameters;(2)Animal experiment: First,the rat ALI model was established by intra-airway nebulized LMWHA and MV,and based on this model,different molecular weight HMWHA(1500 k Da,1600 k Da,1800 k Da)was given by airway atomization in advance,collect lung tissue and bronchoalveolar lavage fluid(BALF)of each group of rats,counted total cells and neutrophils,observe the pathological changes of lung tissue embedded sections after HE staining,use quantitative real-time polymerase chain reaction(q PCR)to detect the m RNA expression of inflammatory factors and chemokines IL-1β,IL-6,IL-8,TNF-α and KC in lung tissue cells,enzyme-linked immunosorbent assay(ELISA)was used to detect the changes in protein expression of IL-1β and IL-6as indicators,to verify the effectiveness of the isolated HMWHA for the prevention and treatment of ALI,and to ensure the feasibility of the separation method used;(3)Cell experiment: Treated with LMWHA for 6 h as the model group,pre-administered HMWHA with different molecular weights as the treatment group,and set up a blank control group.After the completion of the administration,q PCR was also used to detect cytokines and chemokines IL-1β,IL-6,IL-8,TNF-α and KC m RNA expression levels,ELISA detection of IL-1β,IL-6 protein expression levels,to further verify the effectiveness of the product HMWHA and the feasibility of the separation method through in vitro experiments.Result:(1)Compared with the semi-preparation liquid chromatography and high-pressure homogeneity,gel filtration chromatography is stable and can obtain the HMWHA closest to 1600 k Da with the narrowest distribution.Also,this method is stable and repeatable.(2)Compared with the control group,the total number of white blood cells and the number of neutrophils in the BALF group of MV and MV-LMWHA groups increased significantly,the m RNA expression and protein expression of each inflammatory factor and chemokines also increased significantly.The pathological slicing of the lungs showed typical ALI morphological characteristics,among which the upward trend of the MV-LMWHA group was more obvious,and there was a significant difference with the MV group.(3)Compared with the model group of MV+LMWHA,the number of total cells and neutrophils in BALF of rats with different molecular weights in MV+HMWHA group decreased significantly.The pathological slice of the lung shows that the tissue structure of the lungs is clear and complete and the inflamed cell immersion decreases.(4)The m RNA expression of the various inflammatory factors and chemofactors obtained in the cell experiment was consistent with the trend of protein expression and animal experiments.Conclusion:(1)Gel filtration chromatography can be used to separate 1600 k Da HMWHA with narrow distribution from the by-product of 1500 k Da and 1800 k Da HMWHA.The condition parameters of this method are stable and can be further optimized and enlarged linearly according to the need.(2)High ventilation MV can cause lung damage,LMWHA will further induce and aggravate lung inflammation,leading to the occurrence and development of ALI.(3)HMWHA has a preventive therapeutic effect on ALI,which can significantly relieve inflammatory damage in the lungs.In addition to 1600 k Da HMWHA,1500 k Da and 1800 k Da of HMWHA also have an approximate therapeutic effect.It can be assumed that HMWHA in a certain molecular weight range has a similar therapeutic effect on ALI. |
