Effects Of ICS/LABA Combined With Ligustrazine On The Bronchopulmonary Tissue Inflammation Of Chronic Obstructive Pulmonary Disease Rat Models

Objective Rat were exposed to cigarette smoke daily and intratrachealinstillation of lipopolysaccharide (LPS) twice to establish COPD model. Todetect spirometry function and observe the expression of TGF-β1,MMP-9andSLPI in the lung tissue and the CD4/CD8value in blood. Through theSalmeterol/fluticasone(ICS/LABA) and combined with Ligustrazine intervened,observing the change of their expression, To evaluate the effect of differentdoses of ICS/LABA combined with Ligustrazine on the lung inflammation ofchronic obstructive pulmonary disease rat models and its possible mechanism.Methods Seventy Wistar rats were divided into seven groups: the controlgroup(group A)、the model group(group B)、the ligustrazine group(group C)、the middle dose ICS/LABA group(group D)、the middle dose ICS/LABAcombined with ligustrazine group(group E)、 the high dose ICS/LABAgroup(group F)、 the high dose ICS/LABA combined with ligustrazinegroup(group G). Rat were exposed to cigarette smoke daily and intratrachealinstillation of lipopolysaccharide (LPS) twice to establish COPD model.ICS/LABA was inhaled and ligustrazine hydrochloride was intraperitonealinjected, both of them were given to the rats of COPD models for interventionfor30days. The pathological changes in lung tissue were observed andspirometry function was detected. To analyze the number of inflammatory cells in bronchoalveolar lavage fluid(BALF). To observe the collagen deposition onairway by HE. Plasma CD4and CD8levels were determined by flow cytometry.The expression of TCF-βlin BALF was tested by ELISA. The expression ofMMP-9and SLPI in lung was determined by immunohistochemistry. Theexpression of MMP-9mRNA in bronchi and lung was assessed by RT-PCR.Results1. General condition of rats The model group rats activity less, theirhair were dry and oral were purple, their breathe were fast and deeply. Thecontrol group were normal. But after intervention of ICS/LABA andligustrazine, all symptoms above in groups C、D、E、F、G were relief, but groupG was significantly(P<0.05).2. PEF （21.50±1.78） and FEV0.3/FVC（65.27±6.58） in model group were much lower than control group[(41.88±3.26）、(89.63±5.42)]（P＜0.05）；But after intervention of ICS/LABAand ligustrazine, PEF [(25.82±1.96)、(30.62±2.31)、(35.32±2.18)、(33.13±2.62)、(40.12±3.01)] and FEV0.3/FVC [(67.36±4.52)、(80.00±4.56)、(84.80±4.61)、(82.34±5.07)、(87.62±4.76)] in C、D、E、F、G groups weresignificant higher than model group(P<0.05), but group G wassignificantly(P<0.05).3. The total cells in BALF of model group were verysignificantly higher than control group(P<0.05), after intervention, the totalcells in BALF of intervention groups were much lower, the group G was lowerthan other groups(P<0.05). The result of pathology of lung in rats Bronchiallumen stenosis and mucus embolus in model group, and many inflammatorycells in lung that were lymphocytes and macrophages. The alveolar wall wasdamaged, partial of them were formed to bullae and thickening of trachea wall, But after intervention of ICS/LABA and ligustrazine, above symptoms wereless obvious in groups C、D、E、F、G.4. The result of MLI and DI：The MLI and DI in model group[(80.7±16.4)(57.4±16.4)] were higher thancontrol group [(46.2±8.3)、(21.3±4.3)](P<0.05), But after intervention ofICS/LABA and ligustrazine, the MLI [(76.3±14.8)、（71.9±14.6）、(61.2±16.3)、（67.2±15.3）、(54.6±15.7)] and DI [(52.8±5.1)、（47.6±5.7）、(35.2±6.4)、（41.5±5.3）、(29.4±4.8)] in C、D、E、F、G groups were significantly lowerthan model group (P<0.05), but the group G was much better (P<0.05).5.The results of thickness of the airway wall and smooth muscle: The thickness ofthe airway wall and smooth muscle [(36.3±5.8)、(23.7±3.7)] in model groupwere much worse than control group [(17.6±2.6)、(11.8±4.3)](P<0.05), Butafter intervention of ICS/LABA and ligustrazine, the thickness of the airwaywall and smooth muscle in groups C、D、E、F、G were much better than modelgroup(P<0.05), the group G was significant (P<0.05).6. The lever of TGF-β1in BALF：The lever of TGF-β1in model group(138.39±9.94) was higher thancontrol group(52.02±6.37)(P<0.05), after intervention, The lever of TGF-β1inC、D、E、F、G groups [(121.64±11.31)、(108.59±10.56)、(79.64±5.19)、(82.06±8.86)、(59.92±6.51)] were significantly lower than model group(P<0.05), the group G was lower than other groups(P<0.05).7. The resultof CD4/CD8：The lever of CD4/CD8in model group(1.442±0.16)was lowerthan control group(1.809±0.19)(P<0.05), after intervention, The lever ofCD4/CD8in C、D、E、F、G groups [(1.531±0.14)、(1.603±0.25)、(1.673±0.13)、(1.715±0.16)、(1.762±0.17)]were significantly higher than model group (P<0.05), the group G was higher than other groups(P<0.05).8. Byimmunohistochemistry, the expression of MMP-9and SLPI in lung tissue：（1）The expression of MMP-9in model group (3.82±0.31) was much higher thancontrol group(1.80±0.19)(P<0.05), But after intervention of ICS/LABA andligustrazine, the expression of MMP-9in C、D、E、F、G groups[(3.41±0.29)、(3.09±0.28)、(2.73±0.25)、(2.41±0.22)、(2.11±0.23)] were significantly lowerthan model group (P<0.05), the group G was lower than other groups(P<0.05).（2）The expression of SLPI in model group (1.12±0.19) was much lower thancontrol group(3.91±0.32)(P<0.05), But after intervention of ICS/LABA andligustrazine, the expression of SLPI in C、D、E、F、G groups[(1.64±0.22)、(2.11±0.26)、(2.54±0.29)、(2.95±0.28)、(3.45±0.31)]were significantly higherthan model group (P<0.05), the group G was higher than others(P<0.05).9. Theresult of MMP-9mRNA：The lever of MMP-9mRNA in model group(12.440±1.764) was much higher than control group (1.498±0.378)(P<0.05),after intervention, the lever of MMP-9mRNA in C、D、E、F、G groups[(10.785±1.392)、(9.616±1.187)、(6.671±1.026)、(6.156±1.151)、(2.440±0.693)]were lower than model group (P<0.05), the group G was much lower thanothers(P<0.05). The results showed that MMP-9was negatively correlated withFEV0.3/FVC (r=0.8613P<0.01). While SLPI was positively correlated withFEV0.3/FVC(r=0.8321P<0.01). Conclusions1Exposed to cigarettesmoking and intratracheal instillation of lipopolysaccharide can successfullycopy the model of Chronic obstructive pulmonary disease。2Smoking andlung infection could increase the expression of TGF-β1、MMP-9significantly and reduce the expression of SLPI and the level of immunity which might resultin airway remodeling.3Both ICS/LABA and ligustrazine could inhibitedthe inflammation of lung tissues of the COPD rat models which may asscoiatedwith the ICS/LABA and ligustrazine could upregulation of TCF-βland MMP-9expression and elevation of SLPI and enhanced immunomodulatory.4Combined therapy is more efficient than medication alone. And high dose ismore efficient than low dose.