BMSCs-CM Mediates The Regulation Of Endoplasmic Reticulum Stress On Autophagy In Alveolar Macrophages

Objectives 1 Establishment of a silicosis rat model and in vitro dusting cell model confirmed that silica(SiO2)dust can induce the activation of Endoplasmic reticulum stress(ERS).2 To investigate whether the inhibition of SiO2-induced endoplasmic reticulum stress can reduce the level of autophagy during the development of silicosis.3 To investigate whether Bone Marrow Stem Cell Conditioning Medium(BMSCs-CM)can inhibit the occurrence of autophagy by regulating endoplasmic reticulum stress and delay the process of silicosis.Methods This study will be divided into two parts,in vivo and in vitro,and will be studied from the animal and cell fields.1 in vivo experimental animal grouping and model preparation: 45 adult male SD rats were randomly divided into 3 groups: control group,model group,IRE1 inhibitor group,15 in each group.In the model group and the IRE1 inhibitor group,a silicosis rat model was established by injecting SiO2 dust suspension(50 g/L)once with a non-exposed tracheal tube,and the control group was infused with equal amounts of sterile NaCl.The rats in the IRE1 inhibitor group were given STF083010(1.0 mg/kg)by intraperitoneal injection and injected once every 3 days.The remaining two groups were sterilized with equal amounts of NaCl.Each group was killed in batches on days 3,7,and 14 of the model.Detection method: lung tissue hematoxylin-eosin(HE)staining confirmed successful modeling.Morphological changes of alveolar macrophages(AM)were observed by HE staining;immunoblotting was used to detect the expression of IRE1,LC-3 and Beclin-1 protein;immunofluorescence was used to detect the localization of IRE1 and LC-3 protein in AM..2 In vitro experimental animal grouping and model preparation: SPF grade male SD rats about 4 weeks old and weighing about 110 g were selected as animal donors for extracting BMSCs.Another 50 SPF-grade healthy adult male Sprague-Dawley rats were used to obtain AM.The AM was divided into 4 groups: the control group was only added with serum-free medium,and the treatment group was added with SiO2 dust suspension(50 ?g/ml)to establish an in vitro silicosis model.In the IRE1 inhibitor group,40 ?mol/L STF-083010 reagent was added,and in the BMSCs-CM group,an equal volume of prepared conditioned medium was added.Each experimental group was divided into 6h,12 h,18h,24 h,and 36 h after completing the corresponding experimental treatment.The expression of GRP78,IRE1,LC-3 and Beclin-1 was detected by Western blotting.The difference in calculation results was statistically significant with P<0.05.Results 1 morphology observed adherent after the primary BMSCs cells are small,round or oblate,flat or spiral arranged growth,the third generation of BMSCs form tends to be uniform,microscopic observations such as fibroblast-like,nuclear Clear,abundant cytoplasm.2 Results of lung histomorphology: In the control group,the alveolar walls were thin,the structure was clear,and the interstitium was even and translucent.The infiltrating inflammatory cells were seen in the model group at the 3rd day,and there was little serous leakage in the alveolar space.The alveolar septum thickened and the edema was not obvious.At 7 days,the alveolar septum was significantly widened and thickened,with inflammatory infiltrates dominated by alveolar macrophages in the interstitial lung.At 14 days,the alveolar septum was thickened,the structure of the lung tissue was unclear,and a large number of fibrous tissue hyperplasias appeared,with the formation of axillary nodules.Morphological observation of 3 AM: AM cells adhered to the control group were oblate or spherical,and the nucleus structure was clear.Compared with the control group,the AM volume of the silicosis model group increased,the distribution of growth was not uniform,part of the nucleus showed a unilateral distribution,cytoplasm loose,visible spot silica-like crystals.The AM growth of BMSCs-CM group was superior to that of the silicosis model group.Most of the cells had a regular morphology and a clear nucleus structure.4 Western blot results: In vivo results showed that the control group IRE1,LC-3,Beclin-1 protein showed a basic expression,no significant changes in the expression at each time point;silicosis model group compared to the control group protein expression was significantly higher at 7d,14 d At the same time in the control group(P<0.05),it was expressed in increments over time.In vitro results showed that IRE1,GRP78,LC-3,and Beclin-1 proteins were only expressed in the control group,with no significant changes at each time point.The expression level of each protein in the model group increased gradually with time after 6 hours until 18 hours.The peak was higher than that of the control group during the same period(P<0.05).Compared with silicosis model group,the protein expression of IRE1 inhibitor group and BMSCs-CM group was significantly decreased at each time(P<0.05),and the expression of each protein in BMSCs-CM group and IRE1 inhibitor group was not significantly different.similar.The results of immunofluorescence showed that IRE1 and LC-3 were expressed in cytoplasm in vivo.The control group at 3d,7d,and 14 d had only basal expression at different time points.Compared with the same period,the fluorescence of IRE1 and LC-3 in the model group was significantly increased and inhibited.In the agent group,the expression of IRE1 and LC-3 in the model group was decreased,and it changed dynamically with time.Conclusions 1 Silicon dust particles can induce endoplasmic reticulum stress in rat alveolar macrophages.2 IRE1 inhibitor STF083010 can inhibit the ER stress response in alveolar macrophages of silicotic rats and delay the over-activation of autophagy;3 BMSCs-CM can block alveolar macrophages in rats due to endoplasmic reticulum stress The activation of autophagy results in a protective effect on alveolar macrophages in silicotic rats.