Protective Effects Of Kumquat Essential Oil On LPS-induced Pneumonia Mice And BLM-induced Pulmonary Fibrosis In Rats

OBJECTIVE:The aim of this study was to investigate the protective effects of kumquat essential oil in lipopolysaccharide-induced pneumonia mice and bleomycin-induced pulmonary fibrosis rats.METHODS:The ALI model of the protective effect of KEO on LPS-induced pneumonia mice was established.72 male KM mice were randomly divided into vehicle control group,LPS group,dexamethasone(DXMS)group,and KEO(45.5,91).,182 ul·kg-1)group,and were admistrated for 7continuous days(ig),1 hour after the last gavage,except for the vehicle control group,LPS 10 mg/kg was intraperitoneally injected.Organ samples and the blood of the mice was collected after 6 hours,calculated the lung coefficient.The serum liver and kidney function indicators were detected by blood biochemical analysis,the lung tissue cell morphology and pathological changes were observed by HE staining analysis,the positive expression of TNF-? and CX-43 was detected by IHC-P analysis,the serum inflammatory factors of IL-1?,IL-6 and the expression levels of antioxidant enzymes SOD and NO were detected by ELISA analysis.Western blot was used to detect Nrf2,iNOS,COX-2,p-NF-?B p65/NF-?B p65,p-p38 MAPK/p38 MAPK,p-I?B?/I?B? protein expression level.After establishing the PF model of the protective effect of KEO on BLM-induced pulmonary fibrosis in rats,120 male Sprague-Dawley rats were randomly assigned to vehicle control(NS)group,BLM group,prednisone(PDF)group,KEO(In the group of 31.5,63,126 ?l·kg-1),the PF model was induced by intratracheal instillation of BLM(5 mg/kg,injection volume of 1 mL/kg)in the NS group,and the rats were administered intragastrically on the next day.On the 28th day,blood samples and organ samples were collected.FCM detected the expression of CD4+/CD8+ in peripheral blood T cell subsets,blood biochemical detection of serum liver and kidney function indexes,tracheal sections and lung tissue sections were observed by HE and Masson staining analysis,cell morphology and pathological changes were observed to evaluate the degree of pulmonary fibrosis;IHC-P was used to detect the positive expression of TGF-?1;ELISA was used to detect serum inflammatory factors IL-1?,LN,HA and TNF-?,Col-? in lung tissue.The expression level of antioxidant enzyme GPx was detected by Western blotting.The expression levels of IL-10,b-FGF,HMGB1,Caspase3,?-SMA,TGF-?1,TRF2 and p38 MAPK in lung tissue were detected by Western blotting.RESULT:(1)The protective effect of KEO on LPS-induced pneumonia mice:Compared with the Control group,the lung coefficient was increased(P<0.001)and the lung staining of LPS group showed cytoplasmic shrinkage,nuclear irregularity or even fragmentation,separation of adjacent cells and fragmentation of the bridge complex(P<0.001).A large number of inflammatory secretions were observed in the blood vessels,and the levels of ALB and TP were significantly decreased(P<0.01,P<0.05).Compared with the LPS group,the KEO group significantly increased the ALB to the vehicle control group,and the KEO(182 ?l·kg-1)group significantly increased the ALB level(P<0.01)and the TP level(P<0.05);Pathological section images,KEO group significantly improved the histopathological state of acute ALI induced by LPS,of which KEO(91,182 ?l·kg-1)group was the most significant.Stimulation by LPS promoted the expression of inflammatory factors such as IL-6 and IL-1? in serum and lung tissues,and TNF-?,COX-2,iNOS and Nrf2 in lung tissues and promoted p38MAPK and NF-?B.Phosphorylation of I?B? inhibited the expression of SOD and NO in the antioxidant system of the lung;ELISA and IHC-P experiments showed that serum IL-6 and IL-in the KEO(91,182 ?l·kg-1)dose group The positive expressions of TNF-? and CX-43 in 1? and lung tissues were decreased to some extent,and the activity of SOD in the antioxidant system of lungs was increased(P<0.01 compared with LPS group),and the release of NO was decreased(P<0.05).Western Blot experiments showed that KEO(91,182 ?l·kg-1)significantly inhibited the phosphorylation of p38MAPK,NF-?B and I?B? in lung tissue,and COX-2 and iTOS in lung tissue.The expression also showed a significant inhibitory effect and up-regulated the expression level of Nrf2.(2)Protective effect of KEO on BLM-induced pulmonary fibrosis in rats:Compared with NS group,the lung coefficient of rats in BLM group was increased(P<0.05)and tracheal edema was observed by tracheal HE staining in the BLM group,fibroblasts in the intima of the fusiform nucleus,and fibroplasia HE staining of the lung showed a widening of the alveolar septum,cytoplasmic shrinkage,irregular nuclear or even fragmentation,inflammatory cells in some areas,large tears in the central airway and large blood vessels,and a large number of inflammatory cells around,Masson's trichrome staining A large number of densely stained blue collagen fibers were observed;ALT levels in serum were significantly lower(P<0.05).Compared with the BLM group,the KEO group significantly decreased the lung coefficient and increased the ALT level,and the KEO(63,126?l·kg-1)group significantly decreased the lung coefficient level of the day 28 administration group(P<0.05)while increased the ALT level(P<0.01).According to the pathological slice image,the KEO administration group significantly improved the BLM induction.The pathological state of PF prevented pulmonary fibrosis,with KEO(63,126?l·kg-1)being the most significant group,Stimulating by BLM promote the expression of serum IL-1?,HA,LN and lung tissue Col-?,TNF-?,IL-10,b-FGF,HMGB1,Caspase3,a-SMA,TGF-?1,TRF2,p38 MAPK,Inhibition of GPx expression in lung antioxidant system and reduction of CD4+/CD8+ratio;FCM detection showed that KEO group could increase CD4+/CD8+ratio in different degrees compared with BLM group(P<0.01 in 28 days group),ELISA and IHC-P experiment The results showed that KEO(63,126 ?l·kg-1)dose group could significantly inhibit the positive expression of TNF-? and Col-? in serum IL-1?,HA,LN and lung tissues(P<0.05 compared with BLM group).At the same time,the GPx in the lung antioxidant system showed an upward trend(P<0.01 compared with the BLM group).The Western Blot test showed that the KEO(63,126 ?l·kg-1)dose group significantly inhibited IL-10 in the lung tissue.The expression levels of b-FGF,HMGB1,Caspase3,a-SMA,TGF-?1,TRF2,and p38 MAPK protein,wherein the administration group on day 14 was more significant than the administration group on day 28.CONCLUSION:Studies have shown that KEO significantly improves LPS-induced lung injury by blocking p38MAPK,NF-?B and I?Ba pathways to mediate COX-2,iNOS,IL-6,IL-1?,and TNF-?.The expression of CX-43 enhances the activity of antioxidant factor SOD,up-regulates the expression of Nrf2,reduces the release of NO from messenger molecules,and reduces oxidative damage.At the same time,kumquat essential oil can significantly prevent BLM-induced pulmonary fibrosis by inhibiting IL-1?,HA,LN,TNF-?,Col-?,IL-10,b-FGF,HMGB1,Caspase3,a-SMA,TGF-?1 The expression of TRF2 factor reduces collagen deposition,increases the activity of antioxidant enzyme GPx,and enhances the immunity of the body's immune system by increasing the ratio of CD4+/CD8+,thereby counteracting the occurrence and development of pulmonary fibrosis.It is suggested that kumquat essential oil has a good protective effect on lung diseases ALI and PF,and multiple targets and multiple pathways control the various regulatory pathways to reduce lung injury and inhibit pulmonary fibrosis.