Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Establishment and characterization of a fibrotic macrophage-muscle fibrosis model. a) Brightfield image of unstimulated RAW264.7 cells in the top chamber of a RAW-C2C12 coculture system, b) Brightfield image of C2C12 cells in the bottom chamber under basal coculture conditions, c) Fluorescence image of C2C12 cells in the bottom chamber exhibiting myotube morphology, d) Fluorescence image confirming the absence of fibrotic marker expression in C2C12 cells under basal conditions, e) Brightfield image of LPS-activated RAW264.7 cells in the top chamber, f) Brightfield image of C2C12 cells in the bottom chamber displaying a myofibroblast-like morphology post-LPS activation, g) Fluorescence image of phenotypic transition of C2C12 cells to myofibroblast-like cells, h) Fluorescence image demonstrating fibrotic protein expression in LPS-treated C2C12 cells (magenta arrow indicates CD38 cells). Immunostaining images: In panels c) and g), TGF-β, DAPI, and actin are colored red, blue, and green, respectively. In panels d) and h), COL1, α-SMA, CD38, and DAPI are colored red, blue, magenta, and green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Fluorescence, Marker, Expressing, Activation Assay, Immunostaining

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Study of myogenesis through myotube formation. Brightfield images taken using a 10× objective: a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS- H, e) C2C12 RAW LPS - PH, indicating myotube formation, f) Graph representing the number of myotubes. The data points are presented as mean ± SEM (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Immunofluorescence images showing the expression of myogenesis (MyHC) or fibrogenesis (α-SMA and COL) markers. a) C2C12, b) C2C12 RAW, c) C2C12 RAW LPS, d) C2C12 RAW LPS PFD, e) C2C12 RAW LPS - H, f) C2C12 RAW LPS - PH. Semiquantitative expression analysis of the myogenesis/fibrosis markers. g) MyHC/α-SMA and h) MyHC/α-SMA. I) MyHC/α-SMA/DAPI staining, II) MyHC/COL1/DAPI staining. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Gene analysis of fibrogenesis and myogenesis markers. a) COL1, b) MMP8, c) Acta2, d) LOX, and e) MyoG using qPCR. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: AFM images of cells in contact mode in PBS after treatment. a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS - H, e) C2C12 RAW LPS - PH, (f) graph representing nanoindentation measurements denoting the stiffness of the cell. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Mitochondrial metabolism study using a Seahorse extracellular analyzer. Mitochondrial stress test, a) Scheme representing the mitochondrial stress test profile, b) OCR graphs of the inflammation model, c) OCR graphs of the treatment conditions, d) Proton leak measurements, e) ATP production linked to O 2 consumption, f) Maximal respiration, and g) Spare capacity. The data points are presented as mean ± SEM. (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Transmission electron micrographs depicting cell morphology and mitochondrial morphology. (a) C2C12 RAW co-culture with a black box indicating the native muscle cell structure. (b) C2C12 RAW LPS, with a black box highlighting C2C12 phenotypically transitioned to myofibroblast-like cell structures. TEM images of mitochondria within single cells are shown for (c) C2C12 RAW, (d) C2C12 RAW LPS, and (e) C2C12 RAW LPS - PH (PH - Pirfenidone loaded hydrogel) treatment. Yellow arrows point to mitochondria. (The data points are presented as mean ± SEM. (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # and ns indicates significant and non-significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively. Scale bars in figures (a, b) represent 5 μm, c) 1 μm, d, e) 2 μm, and the insets are of a 500 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Transmission Assay, Co-Culture Assay, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: a) Comparative transcriptomic profiles of the control (C2C12 RAW), fibrotic (C2C12 RAW LPS), and treatment (C2C12 RAW LPS - PH) groups, b) Volcano plot of DEGs in the fibrotic (C2C12 RAW LPS) vs control (C2C12 RAW) groups, and c) Volcano plot of DEGs in the treated (C2C12 RAW LPS - PH) vs fibrotic (C2C12 RAW LPS) groups.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: A comparison analysis depicting the a) biological functions and b) canonical pathways associated with fibrosis and healthy muscle regeneration in the control (C2C12 RAW) vs. fibrotic (C2C12 RAW LPS) vs. treatment (C2C12 RAW LPS - PH) groups. The intensity of the heatmap color corresponds to the activation z-score due to the involvement of upregulated and downregulated genes. The supplementary table listing the pathways along with their corresponding z-score values is included in the supplementary section (Table ST1).

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Comparison, Control, Activation Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Principal Component Analysis (PCA) biplots visualise the distinct mechanistic clustering of E. ciliata -derived compounds and reference drugs across all in vitro models. PCA biplots were generated from the combined functional (glucose uptake) and gene expression (qRT-PCR) data for each of the four models: (A) C2C12-Insulin, (B) C2C12-Leptin, (C) C2C12-Adiponectin, and (D) Hepa1c1c7-PA con. Data points (x) represent individual biological replicates, and the ellipses represent the 95% confidence intervals for each treatment group. Loading vectors (black arrows) indicate the direction and magnitude of influence for each variable (e.g. Ptpn1 , Ampka1 , Glucose uptake) on the principal components (PC1 and PC2). The percentage of variance explained by each principal component is shown on the axes. The plots visually confirm the clear separation of the disease control groups (PA-treated) and the distinct mechanistic clusters formed by EC2, EC5, Met, and NAC.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, In Vitro, Generated, Functional Assay, Gene Expression, Quantitative RT-PCR, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Baseline glucose uptake and metabolic gene expression in differentiated C2C12 myotubes under non-PA conditions. Differentiated C2C12 myotubes were treated for 24 h with each of metabolic hormones (insulin, leptin, or adiponectin), reference antidiabetic agents (Met, Duo, NAC), or E. ciliata -derived compounds (EC2–EC5). (A) Glucose uptake (%) was measured in hormone/antidiabetic agent-treated cells (top) and EC-treated cells (bottom). (B) Relative levels of gene expression of Ptpn1 , Pparg , Ampka1 , Ampka2 , and Txnip was assessed via qRT-PCR. ECs were treated at the higher dose (20 μM). The concentration of ECs used in this experiment was 10 μM (L) or 20 μM (H). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. non-treated (vehicle)control. PA , palmitate; Ins-C , insulin- control; Lep-C, leptin-control; Adi-C, Adiponectin-control ; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Pparg , peroxisome proliferator-activated receptor gamma; Ampka1/2 , AMP-activated protein kinase alpha 1/2; Glut4 , glucose transporter 4.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Gene Expression, Derivative Assay, Quantitative RT-PCR, Concentration Assay, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Restoration of the impairment of glucose uptake by E. ciliata -derived compounds in PA-induced insulin-resistant C2C12 myotubes. (A) Glucose uptake levels (%) were quantified. (B) Relative levels of mRNA expression of Ptpn1 , Glut4 , Pi3k , and Akt were analysed via qRT-PCR. EC2-H and EC5-H downregulated Ptpn1 and restored Glut4 levels. ECs were treated at the dose of 20 μM. The concentration of ECs used in this experiment was 10 μM (L) or 20 μM (H). Data are presented as mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01 vs. Insulin + PA control group. † p < 0.05, †† p < 0.01 vs. insulin control group. PA, palmitate; Ins-C, insulin-only control; Ins-PA, insulin + PA; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Glut4 , glucose transporter 4; Pi3k , phosphoinositide 3-kinase; Akt , protein kinase B.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Concentration Assay, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Reversal of leptin resistance by E. ciliata -derived compounds in PA-treated C2C12 myotubes. Glucose uptake in response to leptin stimulation. Glucose uptake was expressed as a percentage of normal cells. (B) Gene expression analysis of Ptpn1 , Jak2 , Stat3 , and Glut4 was performed by qRT-PCR. ECs were treated at the dose of 20 μM. Data are presented as mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01 vs. Lep + PA control group. † p < 0.05, †† p < 0.01 vs. Leptin control group. PA, palmitate; Lep-C, leptin-only control; Lep-PA, leptin + PA; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Jak2 , Janus kinase 2; Stat3 , signal transducer and activator of transcription 3; Glut4 , glucose transporter 4.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Gene Expression, Quantitative RT-PCR, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Effects of E. ciliata -derived compounds on redox homeostasis: NAD⁺/NADH, NADP⁺/NADPH ratios, and FAD levels. C2C12 myotubes were co-treated with palmitate (PA, 300 μM for 48 h) and each test compound or hormone for the final 24 h. The NAD⁺/NADH ratio (A), NADP⁺/NADPH ratio (B), and FAD (C) levels were measured to assess redox homeostasis. Data are presented as mean ± SEM ( n = 5). * p < 0.05, ** p < 0.01 vs. corresponding –PA control group (i.e. basal); † p < 0.05, †† p < 0.01 vs. PA-treated (vehicle) control group. PA, palmitate; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; NAD⁺, nicotinamide adenine dinucleotide (oxidised form); NADH, nicotinamide adenine dinucleotide (reduced form); NADP⁺, nicotinamide adenine dinucleotide phosphate (oxidised form); NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); FAD, flavin adenine dinucleotide.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Control