The Actions Of HMGB1 On Induction Of Myofibrotic Differentiation Following Immune-mediated Injury To Pulmonary Cells And The Intervention Role Of Tianlong-flavonoids Extract

Idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) are two of the most common types of interstitial lung diseases (ILDs), among which IPF has the poorest prognosis, with a median survival of only 3 to 4 years after diagnosis. Although there has been further understanding of the pathogenesis of IPF in recent years, its etiology and exact cellular and molecular mechanisms remain poorly understood. So far, no specifc therapy has been proven unequivocally effective in IPF. The presence of fibroblast foci is one of the most important histopathologic features of IPF. Increased numbers of fibroblastic foci composed of aggregates of activated fibroblasts/myofibroblasts are associated with a disease progression and a worse prognosis in IPF. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle alpha-actin (α-SMA), is believed to be a central event in the process. We have previously identified a 20KDa nuclear protein (GenBank number: BAC34367) that is a C-terminal truncated form of high mobility group Box 1(HMGB1) in the lung of bleomycin-treated mice, with an increased binding toα-SMA promoter. While we found that both the expression ofα-SMA and HMGB1 were significantly increased in lungs of IPF patients and bleomycin-exposed mice. The specific siRNA of HMGB1 could inhibit the expression ofα-SMA and HMGB1 in vivo, thus attenuated bleomycin-induced pulmonary fibrosis in mice. HMGB1, an evolutionarily conserved chromosomal protein, was recently re-discovered to act as a"danger signal"(alarmin) or"damage associated molecular pattern"(DAMP) to alert the innate immune system for the initiation of host defense or tissue repair through binding to pattern recognition receptors (PRRs) widely residing in the cell membrane. Extracellular HMGB1 can be either passively released from damaged/necrotic cells or secreted by activated immune cells. Based on the known function of HMGB1 and our previous findings, we speculated that HMGB1 may be a causal link between lung injury and abnormal repair in the development of IPF. In the present study, we aimed to explore the possibly signaling triggered by extracellular HMGB1 for promoting epithelial-mesenchymal transition(EMT) and myofibroblastic differentiation in the cultured pulmonary epithelial cells(A549) and fibroblasts from NSIP patients and normal. Further more, using the cellular models we tested the role of the flavonoid-extract from a Chinese herbal compound (Tianlong) in treating HMGB1-mediated injury and fibrotic repair. MethodsPart I Preparation of HMGB34367 /HMGB1 recombinant protein P-IEx-9-based eukaryotic expression vector containing cDNA sequences of HMGB34367 or HMGB1, designated as p-IEx-9-HMGB34367 and p-IEx-9-HMGB1 respectively, was successfully constructed and identified. At 48hs after the tranfection of p-IEx-9-HMGB34367 or p-IEx-9-HMGB1 into Sf9 insect cells, the recombinant HMGB34367/HMGB1 was purified with Strep.Tatin affinity chromatography method and identified by immunoblotting.Part II Investigation of HMGB1-triggered signaling for ERK 1/2, Akt, STAT3 and NF-κB in the challenged A549 cells, NLFB and NSIP-LFB and the mediated myofibroblastic differentiation. HMGB1 100ng/ml stimulated A549, NLFB, NSIP-LFB for different times, Westernblot analysis were performed on testing signal pathways consisted of p-ERK 1/2, ERK 1/2, p-Akt, Akt, p-STAT3, STAT3, NF-κB in these cells, andα-SMA expression; Culture supernatants of the challenged cells were collected 24hs after treatment of HMGB1 and stored at -20°C until further analysis by Liquid-chip Assay.Part III The intervention role of Tianlong-flavonoids extracts on the activated ERK 1/2, Akt, STAT3, NF-κB signaling pathways in HMGB1-challenged cells and mediated myofibroblastic differentiation. A549, NLFB and NSIP-LFB, prior to treatment of Tianlong-flavonoids extract at 200μg/ml for 30min, were stimulated with HMGB1 described as above. Westernblot analyses were performed on testing signal pathways andα-SMA expression. Culture supernatants of the challenged cells were analyzed by the same procedures as above.ResultsPart I Preparation of HMGB34367 /HMGB1 recombinant protein. Restriction analysis and sequencing confrimed that plasmids p-IEx-9-HMGB34367 and p-IEx-9-HMGB1 were constructed. Westernblot results demonstrated that the recombinant HMGB34367/HMGB1 was successfully purified from the SF9 cells expressing the recombinants.Part II The action of HMGB1 on activation of ERK 1/2, Akt, STAT3 and NF-κB signaling pathways in A549, NLFB and NSIP-LFB. HMGB1 can significantly up-regulate p-ERK 1/2, ERK 1/2, p-Akt, Akt and NF-κB in the treated A549. Compared to NLFB, NSIP-LFB show abnormal hyper-expression of p-ERK 1/2, ERK 1/2, p-Akt, Akt, p-STAT3, STAT3 and NF-κB, corresponding to hyper-expression ofα-SMA and high levels of IL-6, IL-8 and MCP- 1 under basal conditions; HMGB1 stimulation is capable to induce activation of ERK 1/2, Akt, STAT3 and NF-κB either in NLFB or NSIP-LFB and thereby to increaseα-SMA expression of NLFB in a time-dependent mode and the levels of IL-6, IL-8 and MCP-1. Part III The intervention role of Tianlong- flavonoids extract on the activated ERK 1/2, Akt, STAT3, NF-κB signaling pathways in HMGB1-challenged cells and mediated myofibroblastic differentiation.Treatment of Tianlong-flavonoids extract displays down-regulation of HMGB1-activated Akt, STAT3 and up-regulation of p-ERK in the challenged A549 cells. It can also inhibit abnormal hyper-expression of p-ERK 1/2, p-Akt, p-STAT3, and NF-κB in NSIP-LFBs under basal condition, as well as attenuate HMGB1-stimulated up-regulation of p-ERK 1/2, p-Akt, p-STAT3, NF-κB andα-SMA expression in either NLFB or NSIP-LFB, which correlated with down-regulation of levels of inflammatory cytokines following the intervention.Conclusion1, Extracellular HMGB1 could activate ERK 1/2, Akt, STAT3 and NF-κB signaling pathways in A549, NLFB and NSIP-LFB, thus play important roles in promoting inflammation and fibrosis, It may be a key element of link for inflammation-damage- abnormal repair in the pathogenesis of pulmonary fibrosis;2, Compared to NLFB, NSIP-LFB show abnormal hyper-expression of inflammation and repair signal pathways under basal conditions; extracellular HMGB1 could not only activate the inflammation/abnormal repair signaling in NLFB, but also up-regulate the active signal pathways of NSIP-LFB further. We suggest that the persistent HMGB1 released into the extracellular milieu by necrotic cells or immunocytes induced by immune inflammation had a dominant position in mediating lung fibrosis.3, Tianlong-flavonoids extract has an ability to treat the immune-mediated injury to pulmonary epithelial cells and prevent EMT or fibroblast to myofibroblast differentiation induced by extracellular HMGB1. It also has the capability of correction of abnormal biologic phenotype in NSIP-LFB, thereby to be developed into a new drug for IPF treatment in future.