| Part OneImmunohistochemical pathological and literature review of lung tissue A fatal case caused by paraquat poisoning1 IntroductionParaquat poisoning can induce infiltration of inflammatory cells and a significant inflammatory response in the lungs of patients.Immune activation after paraquat poisoning may be one of the important pathogenic mechanisms.However,there are currently no specific studies on which inflammatory cells can infiltrate into lung tissue.However,there are no relevant studies on which inflammatory cells can infiltrate into lung tissue currently.We dissected a case of dead paraquat poisoning patients.The lung tissue was stained with immunohistochemistry of MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA to determine the type of infiltrating cells.Our study provided a pathological evidence for demonstrating the role of immune activation in paraquat poisoning.2 Method and materialEthics:Sampling and analysis of autopsy was approved by family members and the hospital ethics committee.Medical behavior is in accordance with current Chinese laws and follows the ethics of the Helsinki Declaration.Case report and autopsy:In December 2014,the patient,female,38 years old,was admitted to the hospital because of "oral intake of 100 ml of paraquat for 4 hours".She died after a week of admission.The corpse was kept in a cadaver box with 2-3 ℃ and dissected on the third day after death.Tissue specimens of organs such as heart,lung,kidney are retained.To further clarify the infiltration of immune cells after paraquat poisoning,at 2016-08-12,we sliced the lung tissue wax block again.The lung tissue was performed HE staining and immunohistochemical staining of MPO,CD4,CD8,CD21,CD56,CD68,CD79,SMA.3 Results3.1 HE staining in the lungsTypical histological presentation of this patient involves infiltration of inflammatory cells,diffuse alveolar damage,hyaline membrane formation in alveolar walls and fibrosis.Compared with normal lung tissue,In Fig.1 and Fig.2,there is a disorder and consolidation of lung tissue.Inflammatory cells(mainly neutrophils)are extensively infiltrated into the pulmoriary interstitial and alveolar spaces.In Fig.3 and Fig.4,focal necrosis of the lung tissue was observed,and the normal structure in the necrotic foci disappeared,filling a large number of inflammatory cells(mainly neutrophils),red blood cells,and fibrin deposition In Fig.5,part of the lung tissue showed fibrosis.Fibroblast-like cells with large nucleus and cytoplasm-rich,cellular peripheral fibrosis and inflammatory cell infiltration were seen under the microscope.In Fig.6,alveolar collapse,A large number of alveoli have obvious bleeding.Alveolar filled with pulpy exudate,and contains a lot of cellulose,alveolar epithelial cells shedding and a large number of inflammatory cells infiltration.Significant hyaline membrane formation was seen on the alveolar surface.Pulmonary interstitial edema,thickening of the alveolar wall,and obvious hyperemia and expansion of the alveolar capillaries can also be observed.3.2 NeutrophilsNeutrophils are the most infiltrating inflammatory cells in the lungs.In Fig.8,microscopically,a large number of neutrophils with nucleus deeply stained infiltrated into the pulmonary interstitial and alveolar spacesIn Fig.9,The density of neutrophils adjacent to the blood vessels is significantly increased.Some neutrophils are passing through the vessel wall.3.3 monocytes/macrophagesThe number of monocytes/macrophages infiltrated in the lungs is second to neutrophils.Immunohistochemical Microscopically,a large number of CD68-positive macrophages were distributed in the lung tissue.The macrophages in the hemorrhagic necrosis were significantly increased。The staining of macrophage was uneven.The deep staining was considered to be macrophages that had already phagocytized dust and tissue fragments.The lighter staining consideration was the newly recruited monocytes that had not yet phagocytized the particles.3.4 T helper cellsMarker of immunohistochemistry of T helper cells is generally labeled with antibodies to CD4.In Fig.12,CD4-positive T cells also increased significantly under the microscope,and Th cells deeply stained by the membrane were mostly clustered together in the hemorrhage(Fig.13)and in the pulmonary interstitial(Fig.14).3.5 Cytotoxic T cellThe immunohistochemical CD8 of cytotoxic T cells was positive and increased significantly under the microscope(Fig.15).The cytotoxic T cellsdeeply stained by the membrane were clustered together and distributed in the hemorrhage(Fig.16)and pulmonary interstitial(Figure 17).3.6 Natural killer cellCD56 is a marker molecule for surface expression of NK cells.Microscopically.the number of NK cells was less than the above cells.Deep-stained NK cells were scattered in the pulmonary interstitial(Fig.18,Fig.19).3.7 B cellsCD21 and CD79 are marker molecules on the B cell cell membrane.Microscopically,CD21(Fig.20)and CD79(Fig.21,Fig.22)positive B cell membranes were deeply stained,they were mainly distributed inside the blood vessels and rarely appeared in the pulmonary interstitial.3.8 Fibroblasts/myofibroblastsSMA is the a-actin of smooth muscle,which can be distributed in the cytoplasm of vascular smooth muscle and myofibroblasts.In this patient’s immunohistochemistry,SMA is highly expressed and widely distributed in pulmonary vessels and pulmonary interstitium(Fig.23,Figure 25),We also found high expression of SMA in alveolar capillaries and alveolar walls(Figure 24).In these areas,SMA is usually not expressed.4 ConclusionThrough HE and immunohistocchemical staining,there is a severe inflammatory response in the patient’s lung.Both innate and adaptive immunity are involved in this serious inflammatory response.In innateimmunity,the infiltration of neutrophils and mononuclear macrophages is predominant.In adaptive immunity,Th cells and cytotoxic T cells are significantly increased.The patient’s lung has a severe inflammatory response,both innate and adaptive immunity are involved in this serious inflammatory response..The increase in Th cells and cytotoxic T cells is evident in adaptive immunity.Immunohistochemistry of SMA showed high expression in the lung interstitial cells,alveolar epithelial cells and capillary epithelial cells,which was a tendency to early fibrosis.Part twoExperiment 1:DAMPs,sterile inflammatory pathway and neutrophils involved in paraquat-mediated lung injuryⅠ.IntroductionParaquat poisoning is a thorny toxic disease.Current treatments do not generally improve the mortality.The studys found that the immune system may actively participate in the development and outcome of paraquat poisoning.Our autopsy report and preliminary animal experiment confirmed that innate immunity may play an important role in paraquat poisoning.The immunopathogenic mechanism of paraquat poisoning may be substantially sterile inflammation triggered by DAMPs.However,there is currently no research on the release of DAMPs after paraquat poisoning.Our research on the initiation of the immune response after paraquat poisoning has been innovative.For the above reasons,we propose the following hypothesis in this study:Because a large amount of ROS is produced after paraquat poisoning,the destruction of various cell membrane structures causes cell necrosis and releases a large amount of DAMPs.Sterile inflammatory pathways(the membrane pathway represented by TLR4,the intracellular pathway represented by NLRP3)are activated when DAMPs are combined with PPR.2.MethodThirty-six SD rats were randomly divided into 3 groups:mild group(paraquat 25 mg/Kg intraperitoneal injection),severe group(paraquat 50 mg/Kg intraperitoneal injection)and blank group(normal saline 2 ml intraperitoneal injection),12 in each group.Four rats were taken out at 12.12.72h after intraperitoneal injection of paraquat.After anesthesia,they were sacrificed.The alveolar lavage fluid of the left lung,right lung tissue and blood were collected.Biochemical analyzer detects uric acid(DAMP)in alveolar lavage fluid and serum,alveolar lavage fluid in the left lung and serum cytokines(IL-1β,IL-18,TNF-α,TGFβ1).RNA was extracted from the right upper lobe,and the expression of TLR4,myD88,NF-κB,mtDNA(ND2,DAMP),NLRLP3,ASC,capase-1 and mRNA was detected by qPCR.In order to detect the expression of TLR4,myD88,ASC,capase-1 and NF-κB by Western Blot,proteins in the part of right middle lobe were extracted.Part of the right middle lobe was tested for ROS.Part of the right lower lobe were embedded and sectioned.HE staining was performed for histological lung injury score,(staining + photographing + scoring).Immunohistochemistry was used to detect neutrophils(MPO)and HMGB1(DAMP).Part of the right lower lobe were fixed,embedded and sliced with absolute ethanol.The uric acid was observed by staining Gomori hexamine silver solution.3 Results3.1 Paraquat causes the release of DAMPs.3.1.1 HMGB1Paraquat can cause an increase in HMGB1.Compared with the control group,HMGB1 was significantly increased in different doses at each time point,and increased with time.3.1.2 uric acidParaquat can cause an increase in uric acid.The uric acid in the hexamine silverstaining is brownish black.As the dose and time increase,the area and depth of brownish black are significantly enhanced.In the biochemical test,compared with the control group,the uric acid in the different dose groups at each time point increased significantly,and increased with time.3.1.3mtDNAParaquat can cause an increase in mtDNA.Compared with the control group,the mtDNA of the different dose groups at each time point increased significantly and increased with time.3.2 Activation of the TLR4 pathway3.2.1 Transcription and expression of TLR4Paraquat can result in increased transcription and expression of TLR4.Compared with the control group,the TLR4 of the different dose groups at each time point increased significantly and increased with time.3.2.2 Transcription and expression of MyD88Paraquat can cause an increase in the transcription and expression of MyD88.MyD88 was significantly elevated in the different dose groups at each time point relative to the control group.However,the transcription of MyD88 decreased at 72 hours compared with 24 hours.3.2.3 Transcription and expression of NF-κBParaquat can lead to increased transcription and expression of NF-κB.Compared with the control group,NF-κB was significantly increased in different doses at each time point,and increased with time.3.3 Activation of the NLRP3 pathway3.3.1 Transcription of NLRP3Paraquat can cause transcriptional enhancement of NLRP3.Compared with the control group,NLRP3 in the different dose groups at each time point was significantly increased,and it increased with time.3.3.2 ASC transcription and expressionParaquat can cause increased transcription and expression of ASC.Compared with the control group,the ASC of the different dose groups at each time point increased significantly and increased with time.3.3.3 Caspase-1 transcription and expressionParaquat can lead to increased transcription and expression of Caspase-1.Compared with the control group,Caspase-1 was significantly increased in different doses at different time points,and increased with time.3.4 I L-1β and IL-18 secretionParaquat can cause an increase in the levels of Il-1β and Il-18.Compared with the control group,Il-1β and Il-18 were significantly increased in different doses at different time points(Il-1β only in serum,there was an increasing trend in the mild poisoning group at 12 and 24 hours,no statistical significance)and increased with time.3.5 Secretion of other cytokinesParaquat can cause elevated levels of TNF-α,TGFβ1.Compared with the control group,TNF-α and TGFβ1 were significantly increased in different doses at different time points(only TNF-a in serum,there was no statistically significant increase in the mild group at 24 hours),and increased with time.3.6 Neutrophil count and function3.6.1 Immunohistochemical staining and counting of neutrophils by MPO in lungParaquat can cause an increase in neutrophils in the lungs.Compared with the control group,the number of neutrophils in the different dose groups at each time point increased significantly,and increased with time.3.6.2 ROSParaquat can cause an increase in ROS in the lungs.Compared with the controlgroup,the ROS levels of the different dose groups at each time point were significantly increased,and remained at a high level.There are no significant fluctuations were observed.3.7 Lung pathologyGross observation:The size and weight of the lung tissue in the control group were normal,the lung tissue was pink,and the elasticity was good.The lung tissues inf both the mild and severe groups showed significant swelling and increased weight.The lung tissue is darker in color,harder in texture,and has obvious hemorrhagic foci.The range of bleeding is significantly aggravated with time and dose.Due to the short time,no significant signs of fibrosis were observed.HE staining:con normal;mild and severe groups showed different degrees of pulmonary congestion,edema,inflammatory cell infiltration,fibrosis(obvious at 72 hours).Paraquat can cause an increase in lung damage.Compared with the control group,the lung injury scores of the different dose groups at each time point were significantly increased,and increased with time.3.8 Correlation between neutrophils and lung injury infiltrating the lungThe 72-hour MPO and lung injury scores were analyzed by linear correlation.The results showed that MPO was positively correlated with lung injury score,r=0.908,P<0.01.4 ConclusionIn the early days of paraquat poisoning(within 72 hours),a large amount of DAMPs(HMGB1,uric acid,mtDNA)were released after a large amount of ROS was produced.DAMPs act on PPR on cells and activate a sterile inflammatory pathway(the membrane pathway represented by TLR4-MyD88-NF-κB,the intracellular pathway represented by NLRP3-ASC-Caspasel).Activation of the inflammatory pathway produces the inflammatory factors IL-1β and IL-18,and recruits inflammatory cells dominated by neutrophils to the lungs.Therefore,paraquat mediates sterile inflammation resulting in lung damage.Inflammation caused by paraquat also increases the production of TNF-a and TGFβ1.In the early days of paraquat poisoning,the severity of the inflammatory response(IL-1β,IL-18,TNF-α,TGFβ1,and lung injury scores)and paraquat were somewhat dependent on time and dose.In the early days of paraquat poisoning,the severity of the inflammatory response(IL-1β,IL-18,TNF-α,TGFβ1 and lung injury scores)and paraquat were positively correlated with time and dose to some extent.During the 72-hour period,the number of lung neutrophils was positively correlated with the lung injury score.Part threeExperiment 2:The mechanism of cyclophosphamide on the treatment of paraquat poisoning from the perspective of sterileinflammationⅠ.IntroductionIn Experiment 1,we demonstrated that neutrophils play an important role in paraquat leading to sterile inflammation.The positive feedback effect of DAMPs recruiting neutrophils may be the key to excessive sterile inflammatory responses in paraquat poisoning.Cyclophosphamide is the basic drug for the treatment of paraquat.As early as 1986,E.A DDO reported the use of cyclophosphamide to treat paraquat poisoning.The reason for its treatment is that cyclophosphamide as an immunosuppressive agent can effectively reduce neutrophils and reduce inflammation,and may have a certain therapeutic effect on late fibrosis.Later clinical studies have also found that granulocyte counts in patients with blood routine can be used as prognostic indicators for paraquat poisoning.Whether the mechanism of cyclophosphamide treatment of paraquat poisoning can be elucidated from the standpoint of inhibiting sterile inflammation,currently,there is no relevant research to be available.In our study,We validated the hypothesis proposed by E.A DDO et al.from the perspective of sterile inflammation:Neutrophil deficiency can alleviate the damage caused by paraquat.In this study,rats were pretreated with cyclophosphamide,resulting in an immunosuppressive model of neutropenia.The relationship between cyclophosphamide and sterile inflammation was studied.Differences in inflammatory pathway activation and pulmonary inflammation between the rats in the intervention group and in the exposure group were compared.2.methods and materialsAccording to the results of the preliminary experiment and experiment one,48 SD rats were randomly divided into 4 groups:severely exposed group(paraquat 50 mg/Kg intraperitoneal injection),Intervention group(cyclophosphamide + exposure group,48,24 h before the experiment,100 mg/kg cyclophosphamide intraperitoneal injection,then paraquat 50 mg/Kg intraperitoneal injection),Blank control group(2 ml of saline was injected intraperitoneally),Immunosuppressive group(48,24 h before the experiment,100 mg/kg cyclophosphamide intraperitoneal injection),12 in each group.Four rats were taken out at 12.12.72h after intraperitoneal injection of paraquat.After anesthesia,they were sacrificed.The alveolar lavage fluid of the left lung,right lung tissue and blood were collected.Biochemical analyzer detects uric acid(DAMP)in alveolar lavage fluid and serum,alveolar lavage fluid in the left lung and serum cytokines(IL-1β,IL-18,TNF-α,TGF-β1).RNA was extracted from the right upper lobe,and the expression of TLR4,myD88,NF-κB,mtDNA(ND2,DAMP),NLRLP3,ASC,capase-1 and mRNA was detected by qPCR.In order to detect the expression of TLR4,myD88,ASC,capase-1 and NF-κB by Western Blot,proteins in the part of right middle lobe were extracted.Part of the right middle lobe was tested for ROS.Part of the right lower lobe were embedded and sectioned.HE staining was performed for histological lung injury score,(staining + photographing + scoring).Immunohistochemistry was used to detect neutrophils(MPO)and HMGB1(DAMP).Part of the right lower lobe were fixed,embedded and sliced with absolute ethanol.The uric acid was observed by staining Gomori hexamine silver solution.3.Results3.1.1 HMGB1Immunohistochemical staining:The cells deeply stained with HMGB1 were significantly increased in the exposed group,the intervention group and the cyclophosphamide group,and the increase was most obvious in the exposed group and the intervention group.Quantitative analysis:Compared with the control group,the number of HMBG1-positive cells was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Compared with the exposure group,the intervention group was significantly higher than the exposure group at 24 hours,and the rest did not show significant differences.3.1.2 uric acid Hexamine silver staining in lung tissue:The uric acid with the hexamine silver staining became brownish black.Compared with the control group,the area and depth of the brown-black color in the exposed group,the intervention group and the cyclophosphamide group were significantly enhanced,which was the most obvious in the intervention group.Biochemical analyzerThe performance in serum and alveolar lavage fluid was consistent.Compared with the control group,HMBG1 exposure group,intervention group and cyclophosphamide group were significantly increased,and the intervention group was the most obvious.Compared with the exposed group,the uric acid in the intervention group was significantly higher than that in the exposed group.3.1.3mtDNACompared with the control group,the mtDNA in each time period was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the mtDNA of the intervention group was significantly lower than that of the exposed group.3.2 Effect of cyclophosphamide on the activation of TLR4 pathway mediated by paraquat poisoning3.2.1 Transcription and expression of TLR43.2.1.1 Transcription of TLR4Compared with the control group,the transcription of TLR4 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the TLR4 transcription of the intervention group was significantly lower than that of the exposed group(no statistical difference at 24 hours).3.2.1.2 Expression of TLR4Compared with the control group,the expression of TLR4 was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the TLR4 expression in the intervention group was significantly lower than that in the exposed group(no statistical difference at 72 hours).3.2.2 Transcription and expression of MyD883.2.2.1 Transcription of MyD88Compared with the control group,the transcription of MyD88 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest(no statistical difference at 12 hours in the intervention group).Compared with the exposed group,the MyD88 transcription of the intervention group was significantly smaller than that of the exposed group.3.2.2.2 Expression of MyD88Compared with the control group,the expression of MyD88 was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the expression of MyD88 in the intervention group was lower than that in the exposed group,and there was statistical difference between 12-hours and 24 hours.There was no statistical difference at 72 hours.3.2.3 Transcription and expression of NF-κB3.2.3.1 Transcription of NF-κBCompared with the control group,the transcription of NF-κB in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the NF-κB transcription of the intervention group was significantly smaller than that of the exposed group.3.2.3.2 Expression of NF-κBCompared with the control group,the expression of NF-κB was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the expression of NF-κB in the intervention group was significantly lower than that in the exposed group.3.3 Effect of cyclophosphamide on the activation of NLRP3 pathway mediated by paraquat poisoning3.3.1 Transcription of NLRP3Compared with the control group,the transcription of NLRP3 in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the NLRP3 transcription in the interventiongroup was significantly smaller than that in the exposed group(no statistically significant difference at 72 hours).3.3.2 ASC transcription and expression3.3.2.1 ASC transcription Compared with the control group,the transcription of ASC in each time periodwas significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.And with the extension of time,each group has a tendency to increase.Compared with the exposed group,the ASC transcription of the interventiongroup was significantly smaller than that of the exposed group.3.3.2.2 Expression of ASCCompared with the control group,the expression of ASC was significantly increased in the exposed group,the intervention group and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the exposed group was the second,and the intervention group was the lowest.Compared with the exposed group,the ASC expression of the intervention group was significantly smaller than that of the exposed group.3.4 Effect of cyclophosphamide on downstream inflammatory factors(I L-1β and IL-18)mediated by paraquat poisoning 3.4.11 L-1βIl-1β in serumCompared with the control group,the level of Il-1β was significantly increased in the exposed group at each time period;the intervention group was higher than the control group at 12 hours and 24 hours,and was lower than the control group at 72 hours,but there was no statistical difference.The cyclophosphamide group was lower than the control group at 12 hours and 24 hours,but there was no statistical difference,and the 72 hours were significantly lower,with statistical difference.Compared with the exposed group,the Il-1β level of the intervention group was significantly smaller than that of the exposed group at each time period.Il-1β in alveolar lavage fluidCompared with the control group,the level of Il-1β was significantly increased in the exposed group at each time interval;there was no significant difference between the intervention group and the control group;the level of II-1β was significantly decreased in the cyclophosphamide group.Compared with the exposed group,the expression of Il-1β in the intervention group was significantly lower than that in the exposed group.3.4.2 11-1811-18 in serumCompared with the control group,the level of 11-18 was significantly increased in the exposed group and the intervention group at each time period;the cyclophosphamide group was significantly decreased at 24 hours,and there was an increasing trend at 12 hours and 72 hours,no statistical difference.Compared with the exposure group,the Il-18 level of the intervention group was significantly smaller than that of the exposure group at each time period.I L-18 in alveolar lavage fluidCompared with the control group,the level of Il-18 in the time group was significantly higher in the exposed group;the intervention group was lower than the control group at 12 hours,no statistical difference,and the intervention group was significantly higher than the control group at 24 and 72 hours.The cyclophosphamide group was smaller than the control group,and the decrease of only 12 hours was statistically different.Compared with the exposure group,the IL-18 level of the intervention group was significantly smaller than that of the exposure group at each time period.3.5 Effects of cyclophosphamide on other cytokines(TNF-α and TGF-β)mediated by paraquat poisoningTNF-αTNF-α in serumCompared with the control group,the levels of TNF-α were significantly increased in the exposed group at each time period;the intervention group had an increasing trend,and there was a statistical difference in only 72 hours.the cyclophosphamide group increased at 12 hours,but there was no statistical difference,and the 24 hours and 72 hours were basically the same.There was no statistical difference.Compared with the exposed group,the TNF-a level in the intervention group was significantly lower than that in the exposure group at 12 hours and 24 hours.After 72 hours,the level of TNF-a was significantly higher than that of the exposed group.TNF-α in alveolar lavage fluidCompared with the control group,the levels of TNF-a were significantly increased in the exposed group and the intervention group at each time point;the cyclophosphamide group was slightly lower than the control group,and there was no statistical difference.Compared with the exposed group,the TNF-a level of the intervention group was significantly smaller than that of the exposed group at each time period.TGF-βTGF-p in serumCompared with the control group,the level of TGF-β was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group at each time period.Among them,the exposure group was the highest,the intervention group was the second,and the cyclophosphamide group was the lowest.Compared with the exposure group,the TGF-β level of the intervention group was significantly smaller than that of the exposure group at each time period.TGFβ in alveolar lavage fluidCompared with the control group,the level of TGFP was significantly increased in the exposed group and the intervention group at each time period;the trend of the cyclophosphamide group was not obvious.Compared with the exposed group,the TGFP level of the intervention group was significantly lower than that of the exposed group at each time period.3.6 Effect of cyclophosphamide on the counting and function of neutrophils mediated by paraquat poisoning3.6.1 Immunohistochemical staining and counting of neutrophils in MPO lungImmunohistochemistry:Deeply stained MPO-positive cells were significantly increased in the exposed group and the intervention group,and the cyclophosphamide group was reduced compared with the other groups.Quantitative analysis:Compared with the control group,the proportion of the number of MPO-positive cells in each time period was significantly higher in the exposed group and the intervention group;the cyclophosphamide group was significantly lower than the control group at 12 hours and 24 hours,higher than 72 hours.Control group,but no statistical difference.Compared with the exposure group,the intervention group was significantly lower than the exposure group at each time period.3.6.2 ROSCompared with the control group,ROS in each time period was significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the cyclophosphamide group was the highest,the infected group was the second,and the intervention group was the lowest.Compared with the exposed group,the ROS of the intervention group was significantly smaller than that of the exposed group.3.7 lung pathology scoreGross observation:The size and weight of the lung tissue in the con group were normal,the lung tissue was pink,and the elasticity was good.The lung tissue in the cyclophosphamide group was pale and poorly elastic.Hemorrhagic foci appear in some lung tissues.The lung tissues in the exposed group and the intervention group showed obvious swelling and increased weight.The lung tissue is darker in color,harder in texture,and hasobvious hemorrhagic foci.The range of bleeding increases with time.Compared with the exposed group,the bleeding range of the lung tissue of the intervention group was not uniform,and there was a relatively normal lxrng tissue.The range of residual normal lung tissue gradually decreased with time.Due to the short time,no significant signs of fibrosis were observed.HE staining:normal was normal;the exposed group and the intervention group showed different degrees of pulmonary congestion,edema,inflammatory cell infiltration,and fibrosis(obvious at 72 hours).The inflammatory performance of the intervention group was relatively light compared with the exposure group.Hemorrhagic manifestations were observed in the cyclophosphamide group,and inflammatory cell infiltration was significantly reduced relative to the other groups.Lung injury score:Compared with the control group,the lung injury scores in each time period were significantly increased in the exposed group,the intervention group,and the cyclophosphamide group.Among them,the drug-treated group was the highest,the intervention group was the second^ and the cyclophosphamide group was the lowest.Compared with the exposure group,the lung injury score of the intervention group was significantly smaller than that of the exposure group.3.8 Correlation between neutrophils infiltrating the lung and lung injury pretreated with cyclophosphamideMPO was positive... |
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