Therapeutic Effects Of Palmatine Hydrochloride Against Acute Lung Injury And Pulmonary Fibrosis In Mice

Objectives:To investigate the therapeutic effects of Palmatine hydrochloride(Pal)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and bleomycin(BLM)-induced lung fibrosis(PF)in mice,and to explore its mechanisms of action.Methods:1.One-time intratracheal instillation of LPS and BLM was used to induce ALI model and PF model in mice.2.The lung tissues of mice were weighed to calculate the lung coefficient;conventional HE staining,Masson staining,NF-κB p65 immunohistochemical staining of lung tissues were used to observe the pathomorphological changes of lung tissues under a microscope,and evaluate the degree of inflammation and fibrosis,and the protein expression and location of NF-κB p65.3.The BCA(bicinchoninic acid)kit was used to measure the total protein content in broncho-alveolar lavage fluid(BALF),and ELISA(Enzyme Linked Immunosorbent Assay)was used to detect the content of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-β),interleukin-8(IL-8)and transforming growth factor β1(TGF-β1)in BALF.4.The total number of cells in BALF was counted,and the inflammatory cells were observed by HE staining.5.The commercial kits were used to determine the content of hydroxyproline(Hyp),the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in lung tissues of mice.6.Western blot was used to detecte the protein expression of NF-κB p65,TGF-β1,Smad2/3,JNK,Erk1/2,p38,E-cadherin,Collagen I,α-SMA,and CTGF in lung tissues of mice.Results:1.The lung tissue of ALI mice induced by LPS showed obvious inflammatory reaction,including destruction of alveolar structure,thickening of lung interstitium,infiltration of a large number of inflammatory cells,and the histological inflammatory score was significantly increased.Palmatine hydrochloride significantly alleviated the inflammatory reaction and reduced the inflammatory score in lung tissus of ALI mice.2.The lung coefficient of ALI mice induced by LPS was significantly increased,and Palmatine hydrochloridethe significantly decreased the lung coefficient of ALI mice.3.Total protein concentration,cell number and the levels of inflammatory cytokines including TNF-α,IL-8 and IL-1β in BALF of ALI mice induced by LPS were significantly increased,and these indexes were significantly improved by palmatine hydrochloridethe.4.The protein expression of NF-κB p65 was significantly up-regulated and activated to migrate to the nucleus in the lung tissues of ALI mice induced by LPS,palmatine hydrochloridethe significantly down-regulated the protein expression and inhibited the activation of of NF-κB.5.The body weight of PF mice induced by BLM increased slowly,and the body weight growth rate of high-dose palmatine hydrochloridethe group was significantly higher than that of PF model group.6.The inflammatory reaction in lung tissues of PF mice induced by BLM was obvious on 14th day after modeling,but the deposition of collagen fiber was not obvious.The inflammatory reaction in lung tissue was alleviated on 28th day after modeling,but there was a large amount of collagen fiber deposition,and the content of hydroxyproline in lung tissue was significantly increased.The histological score showed that the scores of alveolitis and fibrosis in PF mice induced by BLM were significantly increased.Palmatine hydrochloride significantly alleviated the inflammatory reaction in the early stage and collagen deposition in the late stage,and reduced the content of hydroxyproline and the scores of alveolitis and fibrosis in PF mice.7.The lung coefficient of PF mice induced by BLM was significantly increased,and the protein expression of NF-κB in lung tissue was significantly up-regulated,the total protein concentration,total cell number and the levels of inflammatory cytokines including TNF-α,TGF-β1and IL-1β in BALF were significantly increased.Palmatine hydrochloride significantly decreased these indexes.8.The activity of endogenous antioxidant SOD in lung tissues of PF mice induced by BLM was significantly decreased,and the content of MDA was significantly increased.Palmatine hydrochloride significantly increased the activity of SOD and decreased the content of MDA in lung tissues of PF mice.9.The portein expression of TGF-β1/Smad signaling pathway related TGF-β1and smad2/3,and MAPK signaling pathway related p38,JNK and Erk1/2,and their downstream CTGF and collagen I were significantly up-regulated in lung tissues of PF mice induced by BLM.Palmatine hydrochloride significantly down-regulated the expression of these proteins.9.The protein expression of epithelial marker E-cadherin was significantly down-regulated,and the protein expression of mesenchymal marker α-SMA was significantly up-regulated in lung tissues of PF mice induced by BLM.Palmatine hydrochloride significantly up-regulated the protein expression of E-cadherin and down-regulated the protein expression of α-SMA in lung tissues of PF mice.Conclusions:1.Palmatine hydrochloride can significantly antagonize the ALI lesions induced by LPS and inhibit inflammatory response in lungs of ALI mice in a dose-dependent manner.2.Palmatine hydrochloride can reduce the protein exudation,total cell number and the levels of TNF-α,IL-8 and IL-1β in BALF,down-regulate the protein expression of NF-κB and inhibit the activation of NF-κB in lung tissues of ALI mice induced by LPS.3.The mechanism of action of Palmatine hydrochloride against ALI is related to inhibiting NF-κB signaling pathway.4.Palmatine hydrochloride can significantly inhibit the inflammation,oxidative stress and alleviate fibrosis in lung tissues of PF mice induced by BLM in a dose-dependent manner.5.The mechanisms of action of Palmatine hydrochloride against PF may include:inhibiting inflammatory response by down-regulating NF-κB protein expression and inhibiting the release of inflammatory cytokines including TNF-α and IL-1β in the downstream of NF-κB signaling pathway,inhibiting oxidative stress by reducing MDA content and increasing SOD activity in lung tissues,inhibiting TGF-β1/Smad and TGF-β1/MAPK signaling pathways by down-regulating the protein expression of Smad2/3,p38,Erk1/2,JNK,CTGF and collagen I,and inhibiting pulmonary epithelial-mesenchymal transition by down-regulating the protein expression of epithelial marker E-Cadherin and up-regulating the protein expression of mesenchymal marker α-SMA,thus intervening the formation of pulmonary fibrosis.