| BackgroundOne-lung ventilation(OLV)refers to the method by which only one lung(non-operative side)is ventilated through a bronchial catheter in patients undergoing thoracic surgery.This is a special ventilation mode used in thoracic surgery,in order to obtain the convenient space and visual field for the operation,but also to reduce the cross infection between the two lungs,but OLV can lead to lung injury.With the prolongation of the time of OLV,lung injury will be aggravated,and may even cause multiple organ dysfunction(MODS).To explore its pathogenesis and effective clinical intervention pathway and to reduce the lung injury caused by OLV will benifit to reduce the incidence and mortality of postoperative complications,and to shorten the hospitalization time of the patients.OLV can cause lung injury of different degree in both lungs,and the mechanism of injury is different.With the conversion of two-lung ventilation mode to one-lung ventilation,the expansion side lung is continually ventilated,its damage is related to the air pressure and tidal volume,the non-ventilatory side lung gradually collapses.Its damage is related to hypoxia,oxidative stress,pulmonary retension and surgical procedures.During one-lung ventilation,a large number of pro-inflammatory cytokines and superoxide ions are produced,followed by severe inflammatory reaction and oxidative stress.Endothelin-1(ET-1)is an important factor in regulating cardiovascular function and is the most potent vasoconstrictor found so far,which is realized by binding with endothelin receptor.Recent studies have shown that ET-1 not only induces vascular fibrosis,stimulates the production of reactive oxygen species,and promotes oxidative stress[7],but also participates in the activation of transcription factor NF-?B and the expression of inflammatory cytokines such as TNF-?,IL-6 and IL-1[8].Theoretically speaking,blocking endothelin receptor can reduce the lung injury induced by ischemia and hypoxia.BQ-123 is a kind of endothelin-1 receptor antagonist.In recent years,more and more studies show that endothelin-1 receptor antagonist BQ-123 has protective effect in heart,brain,lung and other important organs.These results suggest that the protective mechanism may be related to the protection of vascular endothelial cells,the reduction of capillary permeability,the reduction of intravascular exudation,the reduction of inflammatory cell aggregation,the anti-vasoconstriction effect of ET-1,the reduction of intrapulmonary shunt,and the increase of tissue perfusion.The inhibition of lipid peroxidation is related to the improvement of antioxidant capacity.However,There are few studies on the protective effect of BQ-123 on lung injury induced by one-lung ventilation and there is no conclusion about the signal pathway.To investigate the protective effect of BQ-123 on lung injury induced by OLV and its mechanism are helpful for clinical drug research.Phosphatidylinositol 3-kinase serine threonine protein kinase(PI3K-Akt)signaling pathway is a classical signal cascade pathway which can effectively regulate the physiological and pathological mechanism of cells and participate in eukaryotic autophagy value-added apoptosis and so on.The results showed that[4]PI3K-Akt signaling pathway was involved in the protection of BQ-123 against early cerebral injury in subarachnoid hemorrhage.LY294002 is a specific inhibitor of PI3K and can inhibit PI3K-Akt signaling pathway.Hypoxia-inducible factor-la(HIF-la)is a nuclear transcription factor with specific activity under hypoxic condition,an endogenous protective mechanism initiator and a common pathway,and a downstream factor of PI3K-Akt signaling pathway.It was found that it has an important effect on reducing lung injury after ischemia reperfusion[7].The purpose of this study was to investigate the protective effect of BQ-123 on lung injury during OLV in rabbits by establishing a rabbit model of OLV,and to further reveal the mechanism of the protective effect of BQ-123 on lung injury during OLV by detecting the changes of PI3K-Akt signaling pathway and the downstream factor HIF-1?.This research is divided into three parts.Part ?.To establish the animal model of lung injury during OLV ObjectiveTo establish the animal model of lung injury during OLV in rabbits.Methods1.The animal and group:Ten Japanese white rabbits of clean grade were randomly divided into 2 groups(5 rabbits in each group),either male or female,with a body mass of 2.5 ± 0.3 kg.That is Two lung ventilation group(T group),one lung ventilation group(O group),O group will have one lung ventilation for 2 hours,and then have two lung ventilation about 0.5 h,T group will have two lung ventilation for 2.5 hours.2.The establish of one-lung ventilation models in rabbit:After the anesthesia was satisfactory,the right jugular vein was disinfected to establish the venous passage,the left femoral artery was free,and the 24G indwelling catheter was placed in the blood vessel.External pressure gauges are used to detect blood pressure.Tracheotomy was performed.Link the animal ventilator(EMC2002,Beijing Guang Yuan Da Technology Development Co.Ltd.),OLV in rabbits was established bythe parameters of ventilation and the modified single lumen tracheal tube over-deep method proposed by Lan Lan[18].With the left 6th intercostal opening we can see that the left lung changes from inflation to completed collapse,the right lung ventilation model is established successfully.3.The marker for the detection:(1)To measure the W/D ratio in bilateral upper lung tissue.(2)The contents of TNF-? and IL-6 in rabbit lung tissue were measured by ELISA method.(3)The lung tissue of the basal segment of the lower lung was taken for pathological section,and the lung injury score was evaluated by ordinary optical microscope.(4)The femoral arterial blood was drawn at 0.5 ml before OLV(TO),1 hour of OLV(T1),2 hours of OLV(T2)and 30 minutes of TLV(T3).The pH?PaO2?PaCO2?HCO3-and BE were determined by arterial blood gas analysis,and the oxygenation index(OI)was calculated.Referring to the relevant literature reports,this study judged the success of lung injury model making in unilateral lung ventilation from the following three aspects:1.the score of lung pathological injury of lung tissue was more than 6 points,and reached the standard of severe lung injury.2.The value of OI was calculated according to arterial blood gas analysis.When OI<300mm Hg,the lung injury reached the standard of mild ARDS.3.The expression level of cytokines such as TNF-? and IL-6 increased significantly.4.Statistical methods:the statistical software SPSS 20.0 was used to analyze the data.The mean ± standard deviation(x±s)was used to measure the data.The two groups were compared with two independent samples t test.If the data were counted,X2 test was used.The single factor ANOVA of repeated measurement was used to compare the data of different groups in different time periods,if we want to compare the left lung with right lung in one group,we should analyze with paired t-test,P<0.05 is considered signigicant.ResultsThere was significant difference in PaO2 at different time points(F =8.614,P<0.05).Compared with T0,PaO2 decreased significantly(105.8 ± 28.38mmHg?143.80±64.09 mmHg vs 487.14 ±21.48mmHg,P<0.01),and there was significant difference in PaCO2 at different time points(F =5.162,P<0.05).Compared with T0,PaCO2 significantly increased(52.60±5.68mmHg vs 38.80±3.70mmHg,P<0.05),Compared with Tl,the PaCO2 of T3 decreased significantly(36.40 ±6.02mmHg vs 52.60 ± 5.68mmHg,P<0.05),there was significant difference in HCO3-at different time points(F =3.661,P<0.05),and there were marked difference of HCO3-between T1 and T2,T3(23.40±2.60mmol/L vs 19.36± 1.41 mmol/L?17.64±2.18 mmol/L,P<0.05),there were natably difference of pH and BE at different time points(P>0.05).The ratio of lung tissue wet weight/dry weight(W/D):There were significant difference on W/D in both the left lung(t=-14.158,P<0.05)and the right lung(t=-7.220,P<0.05)wihtin group O and group T.The W/D of left lung tissue in group O was higher than that in group T(5.55±0.17 vs 3.93±0.19,P =0.00),The W/D of right lung tissue in group 0 was higher than that in group T(4.76 ± 0.21 vs 3.76±0.23,P =0.00),Comparison of left and right lungs in group O,In group O,the ratio of W/D in the collapsed lung was significantly higher than that in the ventilated lung.(5.55±0.17 vs 4.76±0.21,P<0.01),the differences are statistically significant(t =-5.262,P<0.05).Comparison of left and right lungs in group T,that there was no significant difference in W/D ratio between the left and right lungs(t=-1.351,P>0.05).The content of TNF-? and IL-6 in the lung tissue:Compared to the two groups,there were significant difference on the content of TNF-? in both the left lung(t=-5.955,P =0.00)and the right lung(t=-10.848,P=0.00).The content of TNF-a in left lung tissue in group O was higher than that in group T(37.11±4.03pg/ml vs 19.47±4.30pg/ml,P=0.00),The content of TNF-? in right lung tissue in group O was higher than that in group T(30.39 ± 1.83pg/ml vs 18.38 ± 1.66pg/ml,P =0.00),Comparison of left and right lungs in group O,the content of TNF-a in the collapsed lung was significantly higher than that in the ventilated lung.(37.11±4.03pg/ml vs 30.39±1.3pg/ml,P =0.04),the differences are statistically significant(t=2.749,P<0.05).Comparison of left and right lungs in group T,there was no significant difference on the content of TNF-? between the left and right lungs(t=0.613,P =0.57).Compared to the two groups,there were significant difference on the content of IL-6 in both the left lung(t=-12.063,P =0.00)and the right lung(t=-14.753,P=0.00).The content of IL-6 in left lung tissue in group O was higher than that in group T(20.82 ± 1.33pg/ml vs 7.97 ± 1.98pg/ml,P=0.00),The content of IL-6 in right lung tissue in group O was higher than that in group T(15.94 ± 1.00pg/ml vs 8.38 ± 0.56pg/ml,P =0.00),Comparison of left and right lungs in group O,the content of IL-6 in the collapsed lung was significantly higher than that in the ventilated lung.(20.82 ± 1.33pg/ml vs 15.94 ± 1.00pg/ml,P=0.35),the differences are statistically significant(t =-0.408,P<0.05).Comparison of left and right lungs in group T,there was no significant difference on the content of IL-6 between the left and right lungs(t=38.243,P=0.00).Pathological changes of lung tissue:The lung tissue of T group was normal,the right lung tissue of group O was dark red,and the surface of left lung tissue was mostly crimson,the pathological changes of lung histopathology showed that the lung tissue of T group was normal,and the right lung tissue of group O showed dark red in the right side.There were no interstitial edema,no alveolar edema,neutrophil infiltration,slight interstitial edema in ventilatory side of lung tissue in group O.Mild neutrophil infiltration,mild exudation of erythrocytes in the alveoli,no obvious alveolar edema.Alveolar atrophy,moderate interstitial edema,moderate alveolar edema,moderate alveolar edema,moderate alveolar edema in the collapsed lung tissue Sex granulocyte infiltration,After the analyze with two independent samples t-test,we could find that there were significant different between O Group and T group on both left lung histopathological score(t=-25.913,P =0.00)and right lung histopathological score(t=-6.351,P =0.00),The pathological score of left lung tissue in group O was higher than that in group T(9.40±.55 vs 1.20 ±1.45,P =0.00).The pathological score of right lung in group O was higher than that in group T(3.00±1.12 vs 1.40 ±1.54,P=0.00).By paired t test,the pathological scores of the left and right lung tissues in the group were compared.The difference of left and right lungs in group O was statistically significant(t=29.000,P<0.05).The pathological score of the collapsed lung tissue was higher than that of the ventilated lung tissue(9.40±1.55 vs 3.00±1.12,P=0.00).There was no significant difference between left and right lung pathological scores in group T(t=-1.000,P =0.37).The oxygenation index(OI)The OI of group O was statistically different at different time points(F=217.308,P =0.00).The OI on T1,T2 in O group was significantly lower than that on T0,T3(105.8±28.38mmHg?143.80±64.09mmHg vs 487.14±21.48mmHg?478.20± 22.65mmHg,P<0.05).T group had no significant difference in OI at all time points(F =1.074,P =0.39).Comparison among groups,the OI of O group was significantly lower than that of T group at T 1,T 2(105.8 ± 28.38mmHg?143.80±64.09mmHg vs 499.00± 12.65mmHg?494.00 ± 7.42mmHg,P=0.00).According to the pathological injury score of lung tissue,OI value and TNF-?and IL-6 contents in lung tissue,all the rabbits in the O group reached the standard of lung injury during one lung ventilation.ConlusionRabbits received OLV for 2 hours and TLV for 0.5 hours.The content of TNF-?and IL-6 in bilateral lung tissue was increased to some extent,and the pathological score was increased,the manifestation was more significant in the lollapsed side lung,and the value of OI was decreased,which indicated that the lung had obvious pathological injury in morphology and function.It is an exact animal model of lung injury during OLV.Part ?.The protection of endothelin-1 receptor antagonist against lung injury during one-lung ventilation in rabbitsObjectiveTo observe the protective effect of endothelin receptor-1 antagonist BQ-123 on lung injury during one-lung ventilation in rabbitsMethods1.The animal and group:Fifteen healthy Japanese white rabbits with clean grade were randomly divided into 3 groups,(5 rabbits in each group),that is T group(Two lung ventilation),O group(One lung ventilation)and OB group(OLV with endothelin receptor antagonist BQ-123).2.The establish of one-lung ventilation models in rabbit:It is the same as part I.3.The marker for the detection:(1)To determinate of SOD activity in the lung tissue of the dorsal segment of the lower lobe of both lungs.(2)The contents of TNF-a and IL-6 in lung tissue were measured by ELISA method.(3)The wet and dry weight of bilateral upper lobe lung tissue was measured,and the W/D ratio of lung tissue was calculated(4)The lung tissue was embedded in the basal segment of the lower lobe of the lung,and the lung injury score was evaluated by ordinary optical microscope.(5)Arterial blood gas analysis was performed at T0?T1?T2 and T3,respectively.The oxygenation index(OI=PaO2/FiO2.)was calculated according to the blood gas analysis.4.Statistical methods:the statistical software SPSS 20.0 was used to analyze the data.The mean ±standard deviation(x±s)was used to measure the data.The single factor ANOVA of repeated measurement was used to compare the data of different groups,the comparisons between Pair-wise with the LSD test,if we want to compare the left lung with right lung in one group,we should analyze with paired t-test,P<0.05 is considered signigicant.Results:1.The ratio of lung tissue wet weight/dry weight:There were significant difference on W/D in both the left lung(F =102.168,P=0.00)and the right lung(F =32.755,P =0.00)wihtin group 0,group OB and group T.W/D ratio of left lung tissue in OB group was higher than that in T group(4.53 ±0.18 vs 3.93 ± 0.19,P<0.05),but lower than O.group(5.55 ± 0.17,P<0.05),The W/D ratio of right lung tissue in OB group was also higher than that in T group(4.16 ±0.14 vs 3.76 ± 0.23,P<0.05),but lower than O.group(4.76 ± 0.21,P<0.05).Comparison of left and right lungs in the group,there was no significant difference on W/D ratio between the two lungs in T group(t=1.051,P =0.35).The difference between left and right lungs in O group(t=-40.570,P=0.00)and OB group(t =3.139,P =0.04)were statistically significant.The W/D ratio of the collapsed lung was significantly higher than that of the ventilated lung(P<0.05).2.The lung tissue SOD activityThere were significant difference on the SOD activity in both the left lung(F=36.931,P =0.00)and the right lung(F =55.815,P =0.00)wihtin group O,group OB and group T.The SOD activity of left lung tissue in OB group was lower than that in T group(78.29±1.78U/mg vs 84.42±6.77U/mg,P<0.05),but higher than O.group(62.21 ± 2.10U/mg,P<0.05),The SOD activity of right lung tissue in OB group was also lower than that in T group(82.67 ± 1.54U/mg vs 86.98 ± 1.74U/mg,P<0.05),but higher than O.group(74.21 ±2.44U/mg,P<0.05).Comparison of left and right lungs in the group,there was no significant difference on the SOD activity between the two lungs in T group(t=-0.852,P =0.44).The difference between left and right lungs in O group(t=-7.967,P<0.01)and OB group(t=-3.043,P =0.04)were statistically significant.the SOD activity of the collapsed lung was significantly lower than that of the ventilated lung(P<0.05)3.The content of TNF-? and IL-6 in the lung tissue:There were significant difference on the content of TNF-? in both the left lung(F=24.412,P =0.00)and the right lung(F =71.003,P =0.00)wihtin group O,group OB and group T.The content of TNF-? of left lung tissue in OB group was higher than that in T group(26.30 ± 2.18pg/ml vs 19.47 ± 4.30pg/ml,P<0.05),but lower than O.group(37.11 ± 3.03pg/ml,P<0.05),The content of TNF-a of right lung tissue in OB group was also higher than that in T group(23.12 ± 1.27pg/ml vs 18.38 ±1.66pg/ml,P<0.05),but lower than O.group(30.39 ± 1.83pg/ml,P<0.05).Comparison of left and right lungs in the group,there was no significant difference on the content of TNF-? between the two lungs in T group(t =0.613,P=0.57).The difference between left and right lungs in O group(t=2.749,P<0.05)and OB group(t=3.293,P =0.03)were statistically significant.The content of TNF-a of the collapsed lung was significantly higher than that of the ventilated lung(P<0.05).There were significant difference on the content of IL-6 in both the left lung(F=85.265,P =0.00)and the right lung(F =104.307,P =0.00)wihtin group O,group OB and group T.The content of IL-6 of left lung tissue in OB group was higher than that in T group(14.13 ± 1.26pg/ml vs 7.97 ± 1.98pg/ml,P<0.05),but lower than O.group(20.82 ± 1.33pg/ml,P<0.05),The content of IL-6 of right lung tissue in OB group was also higher than that in T group(11.09±0.89pg/ml vs 8.38 ±0.56pg/ml,P<0.05),but lower than O.group(15.94±1.00pg/ml,P<0.05).Comparison of left and right lungs in the group,The paired t test showed that there was no significant difference on the content of IL-6 between the two lungs in T group(t =-0.408,P=0.70).The difference between left and right lungs in O group(t=5.357,P<0.01)and OB group(t =4.163,P<0.05)were statistically significant.The content of IL-6 of the collapsed lung was significantly higher than that of the ventilated lung(P<0.05).4.Pathological changes of lung tissue:Gross specimens showed normal lung tissue in T group,dark red in right lung tissue and a few bleeding foci in right lung tissue in group O,and deep red in left lung tissue.In the OB group,there were dark red slight bleeding foci in the right lung tissue and normal tissue,and the surface of the collapsed lung tissue was flaky dark red,and the bleeding foci could be seen obviously.No interstitial edema,no alveolar edema,neutrophil infiltration,slight interstitial edema and mild neutrophil infiltration were observed in lung tissue of group T under light microscope.The alveolar atrophy,moderate interstitial edema,moderate alveolar edema,moderate neutrophil infiltration were observed in the collapsed lung tissue.Severe intraalveolar erythrocyte exudation.OB group showed mild interstitial edema,mild neutrophil infiltration,mild intraalveolar hemorrhage,no obvious alveolar edema,and slight interstitial water in the collapsed lung tissue.Swelling,mild neutrophil infiltration,mild alveolar edema,There were significant different in all groups on both left lung histopathological score(F =140.112,P =0.00)and right lung histopathological score(F =20.222,P =0.00).The pathological score of left lung tissue in OB group was significantly higher than that in T group(5.40 ± 1.14 vs 1.20 ± 0.45,P<0.05),but lower than Group O(9.40 ± 0.55,P<0.05).The pathological score of right lung tissue in OB group was also higher than that in T group(2.40±0.48 vs 1.40±0.48,P<0.05),but lower than Group O(3.60±0.47,P<0.05).Comparison of left and right lungs in the group,there was no significant difference on the histopathological score between the two lungs in T group(t=1.000,P =0.37).The difference between left and right lungs in O group(t =29.000,P =0.00)and OB group(t=6.708,P<0.01)were statistically significant.The histopathological score of the collapsed lung was significantly higher than that of the ventilated lung(P<0.05).5.The oxygenation indexThe OI of group OB was statistically different at different time points(F=97.293,P=0.00).The OI on T1,T2 in OB group was significantly lower than that on T0,T3(117.20±41.63mmHg?186.60±70.31 mmHg vs 478.80±24.04mmHg?471.80 ± 48.56mmHg,P<0.05).T group had no significant difference in OI at all time points(F =1.074,P =0.39).Group O has statistical difference in OI at different time points(F=217.308,P=0.00),the OI on T1,T2 in O group was significantly lower than that on T0,T3(105.80 ± 28.38mmHg?43.80 ± 64.09mmHg vs 487.14 ±21.48mmHg?478.20±22.65mmHg,P<0.05).Comparison among groups,the OI of OB group was significantly lower than that of T group at T 1,T 2(117.20 ±41.63mmHg?186.60±70.31mmHg vs 499.00±12.65mmHg?494.00±7.42mmHg,P<0.05).ConlusionAfter the intervention of BQ-123 before OLV,the activity of SOD in lung tissue was increased,the wet/dry ratio was decreased,the content of inflammatory factors such as TNF-a and IL-6 were decreased,and the pathological score was decreased,the manifestation was more significantin in the lollapsed side lung,and the value of OI was decreased,which indicated that endothelin-1 receptor antagonist could reduce lung injury induced by OLV in rabbits.It has protective effect on lung injury of OLV in rabbits.Part ?.Effects of endothelin-1 recepter antagonist on PI3K-Akt-HIF-1?signaling pathway during one-lung ventilation in rabbitsObjectiveTo investigate whether PI3K-Akt-HIF-1? signaling pathway is involved in endothelin-1 receptor antagonist in the process of reducing lung injury induced by OLV and its effect on lung injury in rabbits.Methods1.The animal and group:Twenty healthy Japanese white rabbits with clean grade were randomly divided into 4 groups,(5 rabbits in each group),that is T group(Two lung ventilation),O group(One lung ventilation),OB group(OLV with endothelin receptor antagonist BQ-123)and OBL group((OLV with endothelin receptor antagonist BQ-123 and LY294002).the rabbits in OB and OBL groups were injected with BQ-123(50ug/kg)through femoral vein before OLV.The rabbits in OBL group was given LY294002 0.3 mg/kg by intravenous infusion before BQ-123 for 5 min.Group T maintained 2.5h with TLV,while the rest group received OLV for 2h and TLV for 0.5h.2.The establish of one-lung ventilation models in rabbit:It is the same as part ?3.The marker for the detection:The left upper lung was taken to detect the wet/dry ratio of lung tissue and the left lower lung tissue was taken for pathological section.The lung injury score was observed by ordinary optical microscope,and the expression of phosphorylated Akt and HIF-1? in the left lower lung tissue was detected by Western blot method.4.Statistical methods:The statistical software SPSS 20.0 was used to analyze the statistical data.The mean ± standard deviation was used to measure the data,and the single factor ANOVA of repeated measurement was used to compare the data of different groups,P<0.05 was statistically significant.Results1.The ratio of lung tissuewet weight/dry weight:There were significant difference on the W/D of the left lung wihtin all groups(F=59.768,P =0.00).The W/D of lung tissue in OBL group was higher than that in OB group(5.00 ± 0.25 vs 4.53 ± 0.18,P<0.05),but lower than Group O(5.55 ± 0.17,P<0.05).2.The pathological score of lung tissue:There were significant difference on the pathological score of the left lung wihtin all groups(F=110.571,P =0.00).the pathological score of lung tissue in OBL group was higher than that in OB group(6.60 ± 0.55 vs 5.40 ± 1.14,P<0.05),but lower than Group O(9.40±0.55,P<0.05).3.The expression of p-Akt and HIF-la in lung tissue:The expression level of p-Akt in left lung tissue of group O(0.136 ± 0.038),group OB(0.476 ± 0.081)and group OBL(0.247 ± 0.058)were significantly lower than group T(0.639 ± 0.063).The expression of p-Akt in OBL group was lower than that in OB group and higher than that in O group.There was significant difference between OB group and O group,OBL group(P<0.05).The expression level of HIF-la in left lung tissue of group O(0.076 ±0.016),group OB(0.281 ±0.062)and group OBL(0.129±0.051)were significantly lower than group T(0.569 ± 0.124).The expression of HIF-1? in OBL group was lower than that in OB group and higher than that in O group.There was significant difference between OB group and O group,OBL group(P<0.05).ConlusionAfter the intervention of endothelin-1 receptor antagonist(BQ-123),the expression of p-Akt and HIF-1? in the non-ventilated lung tissue of rabbits was up-regulated,the degree of pulmonary edema was alleviated,and the lung injury score was decreased,which indicated that endothelin-1 receptor antagonist could promote the expression of p-Akt and HIF-1?.After adding LY294002,a specific inhibitor of PI3K,the expression of p-Akt and HIF-1? in non-ventilated lung tissue decreased,pulmonary edema increased and lung injury score increased,indicating that the activation of PI3K-Akt-HIF-1? signaling pathway can reduce lung injury in non-ventilatory side of OLV.However,how endothelin-1 receptor antagonists activate the PI3K-Akt-HIF-1? signaling pathway needs further study. |
PDF Full Text Request