Adropin attenuates pancreatitis‑associated lung injury through PPARγ phosphorylation‑related macrophage polarization

Figure 1. Adropin expression in AP-associated lung injury. (A) Adropin protein expression in serum in patients with AP. (B) Adropin protein expression in serum in the WT + L-Arg group. (C) Serum amylase level in the WT + L-Arg group. (D) Enho mRNA expression in the lung tissue in the WT + L-Arg group. (E) Adropin protein expression in lung tissue in the WT + L-Arg group determined using immunofluorescence. (F) Hematoxylin and eosin staining of pancreatic tissue in the WT + L-Arg group. (G) The adropin protein expression in lung tissue was measured using western blotting. (H) Quantitative analysis of adropin expression determined using immunofluorescence in panel E. (I) Quantitative analysis of the western blots in panel G. *P<0.05 and **P<0.01. AP, acute pancreatitis; L-Arg, L-arginine; NS, normal saline; Enho, energy homeostasis-associated gene.

Figure 2. Results reveal severe acute pancreatitis-associated lung injury in the Adro-KO + L-Arg group. (A) H&E, Masson's staining and immunohistochemistry of lung tissue in the Adro-KO + L-Arg group. (B) Western blot analysis of lung tissue in the Adro-KO + L-Arg group. (C) TUNEL staining of lung tissue in the Adro-KO + L-Arg group. (D) Quantitative analysis of TUNEL staining shown in panel C (n≥8). (E) Quantitative analysis of Masson's staining shown in panel A (n≥7). (F) Quantitative analysis of immunohistochemistry (TGF-β; n≥3). (G) Quantitative analysis of immunohistochemistry (CD68; n≥5). (H) Quantitative analysis of the western blots (caspase-3; n=9). (I) Quantitative analysis of the western blots (PARP1; n=9). *P<0.05 and ***P<0.001. L-Arg, L-arginine; H&E, hematoxylin and eosin; Adro-KO, adropin knockout; NS, normal saline.

Figure 3. Excessive M1 macrophage polarization is observed in the Adro-KO + L-Arg group. (A) Western blot analysis of lung tissue form the Adro-KO + L-Arg group. (B) Co-expression of CD68 and iNOS in lung tissue in L-Arg group determined using immunofluorescence. (C) Co-expression of CD68 and CD206 of lung tissue in the L-Arg group determined using immunofluorescence. (D) iNOS mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (E) CD68 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥7). (F) CD163 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (G) Arg-1 mRNA level in lung tissue from the Adro-KO + L-Arg group (n≥8). (H) Quantitative analysis of immunofluorescence (CD68 + iNOS) staining (n≥5). (I) Quantitative analysis of immunofluorescence (CD68; CD206) staining in macrophages (n≥9). (J) Quantitative analysis of the western blots (PPARγ; n≥6); (K) Quantitative analysis of the western blots (PPARγ Ser273; n≥6). (L) Quantitative analysis of the western blots (PPARγ Ser112; n≥6). *P<0.05. L-Arg, L-arginine; NS, normal saline; Adro-KO, adropin knockout; iNOS, inducible nitric oxide synthase.

Figure 4. Adropin exogenous supplement attenuates severe acute pancreatitis-associated lung injury. (A) Immunohistochemistry of lung tissue from the Adroko + L-Arg + Adro(34−76)group. (B) Western blot analysis of lung tissue from the Adro-KO + L-Arg + Adro(34−76) group. (C) TUNEL staining of lung tissue from the Adro-KO + L-Arg + Adro(34−76) group. (D) Quantitative analysis of immunohistochemistry for MPO (n≥4). (E) Quantitative analysis of immunohistochemistry for CD68 (n≥4). (F) Quantitative analysis of immunohistochemistry for caspase-3 (n≥5). (G) Quantitative analysis of immunohistochemistry for PARP1 (n≥3). (H) Quantitative analysis of the western blots (caspase-3) (n≥5). (I) Quantitative analysis of the western blots (PARP1) (n≥6). (J) Quantitative analysis of TUNEL staining from panel C (n≥5). *P<0.05, **P<0.01 and ***P<0.001. L-Arg, L-arginine; Adro-KO, adropin knockout; NS, normal saline; MPO, myeloperoxidase.

Figure 5. Adropin exogenous supplement induces M2 macrophage polarization. (A) Western blot analysis of lung tissue from the Adro-KO + L-Arg + Adro(34−76) group. (B) Quantitative analysis of the western blots (PPARγ, PPARγ Ser112, PPARγ Ser273) (n≥5). (C) Co-expression of CD68 and iNOS in lung tissue from the Adro-KO + L-Arg+Adro(34−76) group. (D) Co-expression of CD68 and iNOS in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group. (E) Ratio of CD68/DAPI of lung in Adro-KO+ L-Arg+Adro(34−76) group(n≥3); (F) The ratio of iNOS/DAPI in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥3). (G) Ratio of iNOS/CD68 in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥3). (H) Ratio of CD68/DAPI in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥3). (I) Ratio of CD206/DAPI in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥3). (J) Ratio of CD206/CD68 in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥3). (K) CD163 mRNA level in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥6). (L) Arg-1 mRNA level in lung tissue from the Adro-KO + L-Arg + Adro(34−76) group (n≥6). (M) iNOS mRNA level in lung tissue from the Adro-KO + L-Arg +…