Fig 2: Biochemical analysis of tenogenic and repair-related markers in patellar tendons from young and aged rats. (A). Quantitative reverse-transcription polymerase chain reaction was used to determine the gene expression levels of Col1a, TNMD, Scx, COMP, FMOD, MMP3 and TIMP3 in young and aged rat tendons at weeks 2 and 4. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B). Western blot analysis of the protein expression levels of Col-1 and MMP-3. GAPDH was used as an internal control. (C). Quantitative analysis of western blot results. *p < 0.05, **p < 0.01, ***p < 0.001, and n = 3. (D). Comparison of the expression levels of Col-1, Col-3, MMP-1 and TIMP-1 in young and aged rat tendons by immunohistochemical staining. Black scale bar: 20 um. (E). Quantitative analysis of the immunohistochemical staining of Col-1, Col-3, MMP-1 and TIMP-1. *p < 0.05, **p < 0.01, ***p < 0.001, and n = 5

Fig 3: LN attenuates peritoneal adhesion in PPA rats by inhibiting PMC functions. (a) Scanning electron microscope image of LN (×200 nm). (b, c) Adhesion scores and frequency of different groups. Compared with the sham group, *P < 0.05 and **P < 0.01. Compared with the model group, #P < 0.05 and ##P < 0.01. (d) Representative images of adhesion formation, HE, and Masson staining of cecum sections in different groups (100x). (e) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF), migration and adhesion-associated molecules (VCAM-1 and ICAM-1), and ECM-associated molecules (MMP2 and TIMP-1) in different groups. (f–k) qRT-PCR analysis of mRNA levels of profibrotic cytokines (VEGF and CTGF), migration and adhesion-associated molecules (VCAM-1 and ICAM-1), and ECM-associated molecules (MMP2 and TIMP-1) in different groups. Compared with the sham group, *P < 0.05 and **P < 0.01. Compared with the model group, #P < 0.05 and ##P < 0.01. (l) Immunohistochemistry analysis of profibrotic cytokine (CTGF), migration and adhesion-associated molecule (VCAM-1), and ECM-associated molecule (MMP2) in different groups (100x). (m) Immunofluorescence analysis of PPAR? and HIF-1a in different groups (100x).

Fig 4: Ligustrazine suppresses hypoxia-induced PMC functions by activating PPAR?. (a, b) Western blot and qRT-PCR analysis of PPAR? levels in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, *P < 0.05 and **P < 0.01. (c) Immunofluorescence analysis of PPAR? nuclear distribution in hypoxia-induced PMCs treated with 20 µM ligustrazine for 24 h (200x). (d) Flow cytometry analysis of PPAR? nuclear translocation in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. (e) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF) in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. (f, g) FDA staining assay of cell viability in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h (200x). Compared with the control, *P < 0.05 and **P < 0.01. Compared with the ligustrazine group, #P < 0.05 and ##P < 0.01. (h) Western blot analysis of protein expression of migration and adhesion-associated molecules (VCAM-1 and ICAM-1) in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. (i) Western blot analysis of cytoskeleton protein (vinculin) expression in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. (j) Western blot analysis of protein expression of FSP1 in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. (k) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and a-SMA) in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h.

Fig 5: Ligustrazine inhibits hypoxia-induced PMC ECM deposition and EMT transition. (a) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and a-SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. (b–g) qRT-PCR analysis of mRNA levels of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and a-SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, *P < 0.05 and **P < 0.01. (h–k) Immunofluorescence assay of mesothelial-related phenotypic biomarkers (E-cadherin, Cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail, a-SMA) in hypoxia-induced PMCs treated with 20 µM ligustrazine for 24 h (200x).