| Background:Atmospheric fine particulate matter(PM2.5)has been shown to induce lung injury by causing imbalances in the body's pro-inflammatory/ant.i-inflammatory response and studies have shown that the major component of PM2.5 can be induced by Toll-like receptor 4(TLR4).To recognize and trigger signal transduction,in recent years,the TLR4/NF-?B signaling pathway has been recognized as closely related to the pathogenesis of lung injury and its inflammatory response,and it has also been demonstrated that this signaling pathway is involved in PM2.5 induced inflammatory responses.Deng Tietao,a master of Chinese medicine,believes that smog is a sinister poison,and its character is easy to attack thelungs.Its character is damp and easy to brew,which can cause vain,easy to heat from the body,and the pathogenesis of smog induced lung injury.For the four major pathological products of poison,fire,phlegm and phlegm to fight each other in the lungs,the establishment of the law is to clear away heat and detoxify,promote blood stasis and offer the Deng-Shi-Qing-Mai Decoction at the age of 100.This side has been proven in previous clinical studies to reduce the inflammatory response in patients with acute lung injury caused by phlegm and blood stasis.This research was funded by the Guangdong Provincial Administration of Traditional Chinese Medicine Fund Project(No.20193005)and the Guangzhou TCM University Deng Tedtao Fund Project(No.2016030101).In the guidance of the Chinese medicine master Deng Tietao on the academic theory of smog induced lung injury and the early Deng Based on the clinical study of the Deng-Shi-Qing-Mai Decoction in the treatment of acute lung injury caused by phlegm-heat-suppressed lung,we hypothesized that the Deng-Shi-Qing-Mai Decoction with clearing heat-toxicity and blood-activating stasis-removing has a preventive effect on PM2.5 induced lung injury inflammatory response.The mechanism of action is achieved by regulating the TLR4/NF-?B signaling pathway and the release of inflammatory mediators downstream.In order to verify the hypothesis,we conducted a systematic review of the literature and conducted in vivo and in vitro experimental studies to investigate the efficacy and possible mechanism of the Deng-Shi-Qing-Mai Decoction in preventing and treating PM2.5 induced lung injury.The promotion and application in the lay the foundation of research.Objectives:To clarify the preventive and therapeutic effects of the Deng-Shi-Qing-Mai Decoction on inflammatory response induced by PM2.5 in lung injury and to explore its mechanism.Methods:1.Optimization of rat model of lung injury induced by PM2.572 SD rats of 4-6 months old(body weight 200-240g)were randomly divided into 4 groups:control group,PM2.5 low concentration group(0.25mg/mL),PM2.5 medium concentration group(0.5mg/mL),PM2.5 high concentration group(1mg/mL).The animal model was established by unilateral nasal instillation method and each nose was given once on the first day,the seventh day,the 14th day,the 21st day and the 28th day(volume 100 ? L/one)and the third time at 24 hours after the fourth and fifth nasal drops,6 rats were randomly selected from each group,and the abdominal aorta was sacrificed by anesthesia.After each material was taken,the behavioral activities and body weight changes of the rats were recorded.The lung tissue sections were taken and the pathological changes of the lung tissues were observed by HE staining.The intact lung tissues of the left side were taken for the calculation of the ratio of lung dry weight(W/D).The alveolar lavage fluid(BALF)was taken and the lung permeability index was measured by the Brandford method.The blood oxygenation index of the abdominal aorta was measured by a blood oxygen analyzer to evaluate whether the model was successful.2.Prevention and treatment of the Deng-Shi-Qing-Mai Decoction on lung injury inflammation induced by PM2.5The Deng-Shi-Qing-Mai Decoction was made into three different concentration doses of low(0.72g/mL),medium(1.45g/mL)and high(2.90g/mL).72 rats(body weight 200-240g)were randomly divided into 6 groups,namely,control group,model group,dexamethasone group(DXM)and the Deng-Shi-Qing-Mai Decoction low(Low dose),medium dose and high dose groups of 12 each.Except the control group,the other groups were treated with unilateral nasal drip method to establish lung injury rat model with PM2.5 suspension at a concentrat.ion of 0.5mg/mL,which was calculated on the day of modeling and on the first day,respectively.On the 7th,14th,21th and 28th days,each nose was given once a day(volume 100 ? L/head)and from the day of modeling,rats in each Chinese medicine group were given Chinese medicine(3 mL/only),each one day,the dexamethasone group was intragastrically administered with the same volume of dexamethasone solution(concentration 0.02 mg/mL)and the control rats were given the same volume of normal saline daily.Rats in each group were sacrificed by anesthesia after 24 hours after the fifth nasal drop(the 28th day of modeling).Record the behavioral activities,body weightchanges and other general conditions of the rats;take lung tissue sections,HE staining to observe the pathological changes of lung tissue;take the intactlung tissue on the left side,calculate the lung W/D ratio;take alveolar lavage fluid(BALF),The lung permeability index was determined by Brandford method;Giemsa staining was performed for BALF for cell sorting and counting;IL-1?,IL-6,IL-10 and TNF-? were detected by enzyme-linked immunosorhent assay(ELISA)in BALF,inflammatory factor content level;oxygenation index of abdominal aorta blood was measured by blood oxygen analyzer.3.The mechanism of The Deng-Shi-Qing-Mai Decoction on PM2.5 induced lung injury inflammatory responsePreparation of drug-containing serum:24 SD rats(body weight 200-220g)were randomly divided into 2 groups,namely,the drug-administered group(12)and the blank group(12).The Deng-Shi-Qing-Mai Decoction was intragastrically administered with a dose of the Deng-Shi-Qing-Mai Decoction(1.45g/mL)(volume 3 mL/mouse)once a day for 1 week.The blank group was given the same amount of physiological saline in the same manner.Two hours after the last administration,anesthesia was performed by intraperitoneal injection of 2%sodium pentobarbital(2 mL/kg)and blood was taken from the abdominal aorta.The blood was centrifuged,filtered and inactivated and stored for later use.Cell experiment:Rat NR8383 alveolar macrophages were used for in vitro experiments.After cell resuscitation,routine culture and passage were perform-ed and cells in logarithmic growth phase were selected for experiments.The cells were divided into 6 groups,a blank group,a control group,a model group,a Chinese medicine group(Drug)and an inhibitor group(PDTC).The model group,the Chinese medicine group and the inhibitor group were stimulated with PM2.5 suspension at a concentration of 0.5mg/mL and the blank group and the control group were not treated with PM2.5.In the blank group,the model group and the inhibitor group,the cells were cultured in the serum of 20%blank group.The control group and the Chinese medicine group were cultured with 20%rat drug containing serum.After each group was treated for 24 hours,the cells and supernatant were collected Detection.The survival rate of each group was detected by MTT assay.Western blotting was used to detect TLR4,MyD88,IKK a/?,I?B-?,NF-?B p65(cytoplasm and nucleus)on TLR4/NF-?B signaling pathway.Fluorescence immunostaining(IFS)was used to detect changes in NF-?B displacement and nuclear access in cells;gel migration assay(EMSA)was used to detect NF-?B binding to DNA after enucleation;dual luciferase reporter gene was used Luciferase detects NF-?B activation of iNOS and COX-2 gene promoter transcriptional expression;reverse transcription polymerase chain reaction(RT-PCR)and Western blotting to detect post-transcriptional iNOS and COX-2 mRNA expression and iNOS and Expression of COX-2 protein;detection of NF-?B-induced release of inflammatory mediators NO and PGE2 and inflammatory factors IL-10,IL-1?,TNF-? and IL-6 by enzyme-linked immunosorbent assay(ELISA)express the situation.Results:1.Animal model optimization is successful(1)General condition of the rats:In the control group,the low-concentration group and the middle-concentration group,no death occurred.The high-concentration rats died after the first nasal drop and 4 died before the third nasal drop.The fourth nasal anesthesia was given before the nose.One died,and four died after the nose was dropped.Control group:The rats were sensitive to mental reactions,lively and active,with white and shiny coats,normal eating and bowel movements and gradually increased body weight without asking for abnormal breath sounds.Low concentration group:The rats had good mental state,shiny hair and normal eating.After the fourth nasal drop,respiratory symptoms such as asthma,shortness of breath and wheezing appeared and the body weight decreased slightly.Middle concentration group:After the third nasal drop,the rats' mental state was drowsy;the food intake gradually decreased,the weight gain was slow;the fur became yellowish and dull;the symptoms of asthma and shortness of breath appeared and the snoring of the throat was heard in severe cases.High concentration group:The second part of the nose before the nose appeared to be apathetic,shortness of breath,slow response,even claws,cyanosis,some rats after the third nasal drop,wheezing,cyanosis,no eating.The mortality rate is high.(2)Pathological observation and pathological score of lung tissue:pathological observation of lung tissue showed:All the rats in the whole process of the experiment were sensitive,active and active,with white and shiny hair,no abnormal eating and defecation,average weight growth,no abnormal breath sounds;low concentration group:all rats in the whole experiment the mental state is good,the hair is shiny,the eating is normal and the weight of some rats is slightly decreased in the late stage;most rats have a laryngeal snoring after the fourth nasal drop;the middle concentration group:the majority of the rats are dripped in the fourth time.After the emergence of mental wilting,the fur gradually yellowed and dull,the food intake gradually decreased,the weight gain was slow,occasionally mild ast.hma symptoms,severe cases can hear the snoring of the throat;high concentration group:some rats in the second drop There was a feeling of wilting in the nose,shortness of breath,slow response,cleftnails and cleft lip;most rats developed wheezing,cyanosis,no eating and high mortality after the third nasal drop.Lung histopathology score:After the first dose,the high concentration group was signi ficantly higher than the control group compared with the control group;the low concentration group and the middle concentration group had no significant difference compared with the control group.After the second sampling,the high concentration group was significantly higher than the control group compared with the control group;the low concentration group and the middle concentration group had no significant difference compared with the control group.After the third sampling,the middle concentration group was significantly higher than the control group compared with the control group;the low concentration group had no significant difference compared with the control group.(3)Laboratory indicators:After the first sampling:the low concentration group and the middle-concentration group had no significant difference in lung permeability index,lung W/D ratio and oxygenation index compared with the control group(P>0.05);Compared with the control group,the lung permeability index and lung W/D ratio were significantly increased and the oxygenation index was significantly decreased(P<0.01).After the second extraction:the low-concentration group and the middle-concentration group had no significant difference in lung permeability index and lung W/D ratio compared.with the control group(P>0.05);the middle concentration group compared with the control group,oxygen The index of the combination decreased significantly(P<0.01).Compared with the control group,the lung permeability index and lung W/D ratio increased significantly,and the oxygenation index decreased significantly.The difference was statistically significant(P<0.05).After the third extraction:the lung permeability index and lung W/D ratio were not significantly different in the low concentration group compared with the control group(P>0.05);the lung permeability index was compared in the middle concentration group compared with the control group.The lung W/D ratio increased significantly,the oxygenation index decreased significantly and the difference was statistically significant(P<0.01).The rats in the high concentration group died before the third extraction,which affected the detection.2.The Deng-Shi-Qing-Mai Decoction has a preventive effecton PM2.5 induced lung injury inflammatory response(1)General condition of rats:No death was observed in each group of rats.The rats in each group were observed as follows:Control group:All rats were agile,energetic,lively and joyful,with white and shiny hair,no abnormalities in eating and stool,no abnormal breath sounds and throat humming sounds.Model group:Most rats have slow activity,like static and stagnation,wilting,yellow hair and asthma symptoms.In severe cases,they can hear snoring in the throat;most rats eat in the whole process of modeling gradually decreases.dexamethasone group:Most of the rats were sedative,mentally exhausted,slow-moving,yellow-white and yellow and a small number of rats developed shortness of breath.Some of them smelled snoring in the throat.Most of the modeling process was large.The amount of rat feeding decreased significantly in the later stage.Each Chinese medicine treatment group:the vast majority of rats were active,energetic,flexible,moderate in food intake,shiny in hair and did not smell snoring in the throat;most rats in the low-dose group showed cough,short.ness of breath,etc.Symptoms;a small number of rats in the middle and high dose groups developed cough,shortness of breath and mild symptoms.Rat body weight:1d body weight:There was no significant difference in body weight between the groups.Body weight of the 7th day:the model group,the dexamethasone group and the low dose group had a significant decrease in body weight compared with the control group(P<0.01).There was no significant difference in the body weight between the middle and high dose groups compared with the control group(P>0.05).On the 14th day,there was no significant difference in body weight between the dexamethasone group and the low-dose group(P>0.05).There was no significant difference in the body weight between the middle and high dose groups compared with the control group(P>0.05).Body weight of the 21st day:Compared with the control group,the body weight of the model group decreased significantly(P<0.01);the weight of the rats in the middle and high dose groups was significantly higher than that of the model group(P<0.01);dexamethasone Compared with the model group,there was no significant difference in body weight between the two groups(P>0.05).The weight of the rats in the middle and high dose groups was signi ficantly higher than that in the dexamet hasone'group(P<0.01).The middle and high dose groups Compared with the control group,there was no significant difference in body weight between the two groups(P>0.05).Body weight at 28d:Compared with the control group,the body weight of the model group and the dexamethasone group was signi ficantly lower(P<0.01).Compared with the model group,the body weight of the low,medium and high dose groups was significantly higher(P<0.01);the low,middle and high dose groups had signi ficantly higher body weight than the dexamethasone group(P<0.01);the low,medium and high dose groups compared with the control group,the rat body weight There,was no significant difference in values(P>0.05).(2)Pathology of lung tissue:The control group:the alveolar structure was normal,the distribution was uniform,the alveolar cavity was full and smooth,no abnormal increase,the alveolar wall was free of congestion and edema,fusion and rupture,the alveolar septum was not thickened and widened;a very small amount of oozing and inflammatory cells appeared in the interstitial lung.The bronchial wall was free of congestion and stenosis;the model group:the whole lung tissue was severely damaged and a large number of inflammatory cells exuded,congested and edema,no intact alveolar structure;bronchial wall congestion and thickening;dexamethasone group:visual field A small amount of inflammatory cells and hemorrhage and edema in the lung tissue;more complete alveolar structure,a small part of the alveolar cavity collapse,a small amount of alveolar fusion,rupture;partial pulmonary interstitial congestion and inflammatory exudation;bronchial wall slightly thickened;low Dosage group:Inflammatory exudation and hyperemia of lung tissue in the visual field;most alveolar structures are incomplete,alveolar cavity collapses,alveolar septum fusion,thickening;pulmonary interstitial hyperemia with inflammatory cells increased;bronchial wall thickening,hyperemia;High-dose group:the lung tissue structure in the visual field is relatively intact,a small amount of alveolar cavity collapses or increases and the alveolar wall has a little edema,fracture and thickening;A small amount of interstitial inflammatory cell infiltration;no significant congestion bronchial wall,narrow.Morphology and pathology of lung tissue:Morphological observation of lung tissue:Control group:The surface of lung tissue was smooth,the lungs were evenly pale pink and no swelling,congestion,exudation and obvious infarct were observed.Model group:bilateral lung tissue was swollen with irregular fine sand grain,dark red color,more hemorrhagic lesions under the lung capsule,and even flaky.In the dexamethasone group,the surface of the lung tissue was smooth,and the whole lung was pale pink with a spotted bleeding point and a small amount of exudate.In the low-dose group,the volume of both lungs increased significantly and the center of the lung tissue was dark red and bloody and the periphery was pale pink.In the middle and high dose groups,the volume of the lungs did not increase significantly and the whole was pale pink.No obvious hyperemia and exudate were observed.(3)Laboratory indicators:After successful establishment of the animal model,the expression of inflammatory factors IL-1?,IL-10,TNF-?,IL-6,lung W/D ratio,lung permeability index and inflammatory cells in BALF compared with the control group.The number of patients increased significantly and the oxygenation index decreased significantly.The difference was statistically significantv(P<0.01).The low dose group compared with the model group,The expression of the inflammatory factors IL-1?,IL-10,TNF-?,IL-6 in BALF,lung W/D ratio,lung permeability index and inflammatory cell number decreased to some extent,oxygenation index increased to some extent;medium and high dose group compared with model group,BALF inflammatory factor the expressions of IL-1?,IL-10,TNF-?,IL-6,lung W/D ratio,lung permeability index and number of inflammatory cells decreased and oxygenation index increased.The difference was statistically significant(P<0.05).Compared with the dexamethasone group,the therapeutic effect was not significantly different between the dexamethasone group and the dexamethasone group(P>0.05),but the Chinese medicine effect was significantly better than dexamethasone in reducing the symptoms of lung injury.Dexamethasone is superior to traditional Chinese medicine in controlling the release of inflammatory factors.3.The Deng-Shi-Qing-Mai Decoction can alleviate PM2.5-induced inflammatory response by regulating TLR4/NF-?B signaling pathway and its downstream inflammatory mediators(1)The effect of different concentrations of PM2.5 on the survival rate of NR8383 cells:Compared with PM2.50.5mg·mL group,the cell survival rate of PM2.5mg mL group decreased significantly at 12h,24h,48h,72h;and PM2.50.5mg mL Compared with the group,the cell survival rate of PM2.54mg mL group decreased significantly at 12h,24h,48h,72h.Compared wi th the PM2.50.5mg mL group for 24h,the cell survival rate of 72h decreased significantly,the difference was stat i stically significant(P<0.01).(2)Effects of each group on related proteins in TLR4/NF-?B signaling pathway:TLR4,MyD88,IKK?/?,I?B-?,nuclear NF-?B p65,intracellular NF-K B p65 protein in the control group compared with the blank group.There was no significant di fference(P>0.05).Compared with the blank group,TLR4,MvD88,IKK ?/?,nuclear NF-?B p65 protein expression increased,I?B-?,intracellular NF-?B p65 protein expression decreased,the difference was statistically significant.Significance(P<0.05);Compared with the model group,TLR4,MyD88,IKK ?/?,nuclear NF-?B p65 protein expression,I?B-?,intracellular NF-?B p65 protein expression,the difference was statistically significant(P<0.05);Chinese medicine group Compared with the PDTC group,there was no significant difference in the expression of the above proteins(P>0.05).(3)Effects of each group on NF-?B translocation and nuclear access:Compared with the blank group,the control group and the blank group showed high intensity red fluorescence in the cytoplasm,weak fluorescence in the nucleus and colocalization of NF-?B p65 protein with the nucleus.There was no significant difference in regional fluorescence intensity values(P>0.05).Compared with the blank group,the fluorescence intensity of the whole cells increased,the fluorescence aggregation in the nucleus increased significantly and the fluorescence intensity values of NF-?B p65 protein and nuclear colocalization region were significantly enhanced.The difference was statistically significant(P<0.01).Compared with the model group,the intracellular fluorescence intensity decreased,the fluorescence aggregation in the nucleus was significantly less and the fluorescence intensity value of NF-K B p65 protein and nuclear colocalization region was significantly lower.The difference was statistically significant(P<0.01).There was no significant difference in the intensity of fluorescence intensity between the two groups(P>0.05).(4)Effects of NF-?B on DNA binding activity in each group:There was no difference in the binding of NF-KB to probes in the control group compared with the blank group(P>0.05).Compared with the blank group,NF-KB and NF-?B were compared with the blank group,The binding ability of the probe was enhanced and the gray value was significantly increased.The difference was statistically significant(P<0.01).Compared with the model group,the binding ability of NF-?B to the probe was weak and the gray value was significantly reduced.The difference was statistically significant(P<0.01);There was no significant difference in gray value between Chinese medicine group and PDTC group(P>0.05).(5)Effects of PM2.5-treated cells on NF-K B transcriptional activity:Compared with the other three groups(group A,group B and group C),the relative fluorescence intensity values in cells were significantly increased.The difference was statistically significant(P<0.01).(6)Effects of NF-x B on post-transcriptional activation of inflammatory mediators:There was no significant difference in the relative expression of iNOS and COX-2 mRNA and protein,NO and PGE2 between the control group and the blank group(P>0.05).Compared with the blank group,the relative expression levels of iNOS and COX-2 mRNA and protein,NO and PGE2 were significantly increased(P<0.01).Compared with the model group,iNOS and COX-2 mRNA and protein were compared with the model group.The relative expressions of NO and PGE2 were significantly decreased and the difference was statistically significant(P<0.05).There was no significant difference in the relative expression of iNOS and COX-2 mRNA and protein,NO and PGE2 between the Chinese medicine group and the PDTC group(P>0.05).(7)The effect of each group on the release of NF-?B to promote inflammatory factors:There was no significant difference in IL-1?,IL-6,IL-10 and TNF-? between the control group and the blank group(P>0.05);Compared with the blank group,the levels of IL-1?,IL-6,IL-10 and TNF-? were significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,IL-1? and IL-6 were compared between the Chinese medicine group and the model group.The levels of IL-10 and TNF-? were significantly decreased(P<0.01).There was no significant difference in the content of inflammatory factors between the Chinese medicine group and the PDTC group(P<0.01).Conclusion:1.Using the PM2.5 suspension with a concentration of 0.5mg/mL and avolume of 100 ? L,the main features of the lung injury animal model can be successfully optimized by intranasal drip infusion 5 times(28 days after modeling).PM2.5 induced lung injury rat model in accordance with the mechanism of human lung injury.2.After PM2.5 is exposed to lung tissue,it can cause lung injury symptoms in rats,pathological damage of lung tissue and inflammat ion.The prevent ion and treatment of the Deng-Shi-Qing-Mai Decoction on PM2.5 induced lung injury inflammatory response is manifested in the relief of lung injury symptoms,the reduction of pathological damage in lung tissue and the reduction of inflammatory response and there is a dose-effect relationship.3.After PM2.5 siimulates cells,it can interfere with the expression of related proteins in TLR4/NF-?B signaling pathway,activate NF-?B to shift and enter into nucleus,promote binding with target DNA and initiate transcription,causing a large number of inflammatory mediators.The formation of inflammatory factors and the development of inflammatory reactions;The Deng-Shi-Qing-Mai Decoction can reduce the activation of NF-?B,reduce the activation of NF-?B and reduce the binding activity to targetDNA by regulating the expression of related proteins in the TLR4/NF-?B signaling pathway.It reduces the production of inflammatory mediators and the release of inflammatory factors,thereby reduceing the inflammatory response. |
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