Bone Marrow Origin Of Myofibroblasts In The Paraquat Caused Lung Injury

Backgroud and objective: Paraquat is highly toxic for human and animals. When intoxicated by oral administration, paraquat was absorbed and concentrated in the lung. Paraquat can cause acute pulmonary inflammation and develop to irreversibility pulmonary interstitial fibrosis, leading to death. Myofibroblast can both excret cytokine and regular the deposition of extracellularis matrix (ECM), which plays a key role in the development and progression of the pulmonary fibrosis(PF). It is not clear now about the origin of the myofibroblast. We try to identify whether the myofibroblast origins from the bone marrow and plays a role in the lung injury induced by paraquat intoxication, and aim to offer theoretic base for clinical therapy.Methods. The circulating fibroblasts of inbreeding line male f344 rats were separated by density gradient centrifugation and cultured for 10～12 days. The circulating fibroblast was labeled by DAPI and its vigor was detectd. 30 inbreeding line male t344 rats were randomized to 2 groups. The paraquat intoxication model group (group PQ): the rats were given a single dose 80mg/kg of paraquat by intragastric administration, and then were given circulating fibroblast labeled by DAPI by caudal vein injection. The control group (group NS): the control rats were given equivalent volume of normal saline, and then circulating fibroblast. The rats were sacrificed at 1 st, 7th and 14th days post exposure, 5 rats every time, then the lung specimens were obtained. Lung specimens were fixed for HE stain to evaluate the area of alveolar septum. The intensity of expression ofα-SMA and CD³⁴ was detected by immunohistochemistry. The expression of DAPI was detected by immtmofluorescence.Results:â‘ The circulating fibroblast was separated successfully and the proportion of living cell was over 90%.â‘¡Progressive diffuse airsacculitis, with alveolar dropsy, pulmonary hemorrhage, hyaline membrance and inflammatory cell infiltration was found in the lung of PQ intoxicated rats. Compared with control group, the area of alveolar septum in paraquat intoxication model group was significantly lager at the 1 st, 7th and 14th days post exposure(P=0.000; P=0.000; P=0.046).â‘¢The intensity of expression ofα-SMA in paraquat intoxication model group was significantly higher at the 1st, 7th and 14th days post exposure than that in control group(P=0.012; P=0.000; P=0.000). The intensity of expression ofα-SMA in paraquat intoxication model group at the three time points augmented by degrees, a significant difference was found among them(P=0.009).â‘£Compared with control group, the intensity of expression of CD34 in paraquat intoxication model group was higher at the 1st, 7th and 14th days post exposure, but no significant difference was found among them(P=0.446; P=0.326; P=0.594). No significant difference was found in the intensity of expression of CD34 of paraquat intoxication model group among the lst,7th and 14th days post exposure(P = 0.757).⑤Bright blue fluorescence was detected by immunofluorescence. The circulating fibroblast in paraquat intoxication model group was only detected surrounding the blood vessel at the 1 st day post exposure, which was detected both in the lung interstitium and surrounding the blood vessel at the 7th and 14th days post exposure.While little blue fluorescence can be detected in control group.Conclusions: The rat model of lung injury induced by paraquat can be successfully prepared by given a single dose 80mg/kg of paraquat by intragastric administration. The way and dose of paraquat administration is suitable. Paraquat intoxication can cause lung injury in rat model, which develops to alveolitis and pulmonary interstitial fibrosis. The circulating fibroblast can be separated by density gradient centrifugation, cultured in vitro and used to transfusion successfully. The DAPI labeling is effective and stable. The blue fluorescence of DAPI can be detected in two weeks after cell transfusion. The upregulation of pulmonary expression ofα-SMA in paraquat intoxication rats indicates the presention of myofibroblast in the lung. The expression of blue fluorescence of transfued cell labeled by DAPI indicates that the circulating fibroblast plays a role in the reparation of lung injury induced by paraquat intoxication.