Journal: Frontiers in Pharmacology

Article Title: Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-κB and regulating protein synthesis/degradation

doi: 10.3389/fphar.2022.1056460

Figure Lengend Snippet: TNFα, associated with grip strength in population, increases in atrophied muscle induced by denervation and induces muscle atrophy in vivo and in vitro . (A) Genetic association study analysis for grip strength to find SNPs with genome-wide significant association for right-hand grip strength. There was a novel loci, and the SNP rs2857702 ( p -value = 1.411e-15), represented with purple rhombus, was genome-wide significantly associated with right-hand grip strength and was close to TNF (synonyms : Tnfα ) gene. (B–D) 2-month-old male C57BL/6 sham and denervated (Den) mice were harvested on day 12 after surgery. n = 3–6/group. (B–C) TNFα mRNA levels (B) and protein levels (C) in gastrocnemius muscles. (D) body weight (BW), Lean weights of the tibialis anterior (TA), extensor digitorum longus (EDL), soleus, and gastrocnemius (Gas) muscles. (E–F) Effects of indicated different concentrations of TNFα on myotube formation from C2C12 cells (E) and primary myogenic cells (MCs; CD45 − ; CD31 − ; CD11b − ; Sca1 − ) (F) isolated from skeletal muscles of 3-month-old C57BL/6 mice. No. of MyHC-positive myotube/area was measured. n = 3/group; * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: For primary myogenic cells and C2C12 cells differentiated myotube staining, primary antibody to MyHC (1:100, R&D Systems, Minneapolis, MN, United States, catalog MAB4470) was incubated at 4°C overnight.

Techniques: In Vivo, In Vitro, Genome Wide, Muscles, Isolation

Journal: Frontiers in Pharmacology

Article Title: Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-κB and regulating protein synthesis/degradation

doi: 10.3389/fphar.2022.1056460

Figure Lengend Snippet: Morroniside inhibits proteasomal and autophagic degradation pathways in C2C12 myotubes treated with TNFα and/or muscles from denervated mice. (A–C, F–J) C2C12 cells were treated with TNFα (10 ng/ml) and administrated with or without morroniside (Mor, 80 or 160 μg/ml), then the cells were induced to differentiation. n = 3 samples/group. (A–B) Follistatin, Myostatin, Bmp11, Alk4, Alk5, Alk7, Smad7, Smad2, Smad3 (A) , Foxo3, Atrogin1 and MuRF1 (B) mRNA levels in C2C12 myotubes were measured by qPCR. (C) Atrogin1 and MuRF1 protein levels in C2C12 myotubes were measured by WB and quantified by Image J. n = 3 samples/group. (D–E and G–I) 2-month-old male C57BL/6 mice were randomly divided into Sham operation, Denervation (Den), Den + morroniside (Mor, 20 μg/kg) groups based on body weights, and received sham operation or sciatic nerve transection and were treated with Veh or morroniside (Mor) 1 x/day, starting 1 day after operation for 12 cycles. n = 6/group. (D–E) Atrogin1 and MuRF1 mRNA (D) and protein levels (E) in gastrocnemius muscles were measured by qPCR and WB, respectively. Quantification of Atrogin1 and MuRF1 protein levels by Image J. n = 6 samples/group. (F) MyHC protein levels in the cell lysates were measured by WB and quantified by Image J. T + L = TNFα +80 μg/ml Mor; T + H = TNFα +160 μg/ml Mor. n = 3 samples/group. (G) MyHC protein levels in the gastrocnemius lysates were measured by WB and quantified by ImageJ n = 6 samples/group, * p < 0.05; ** p < 0.01. (H) WB of GAPDH and MyHC ubiquitination after IP with anti-MyHC antibody and IB with anti-Ub antibody in gastrocnemius lysates. (I) Bnip3, Lc3a, Lc3b mRNA levels in C2C12 myotubes were measured by qPCR. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For primary myogenic cells and C2C12 cells differentiated myotube staining, primary antibody to MyHC (1:100, R&D Systems, Minneapolis, MN, United States, catalog MAB4470) was incubated at 4°C overnight.

Techniques: Muscles, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-κB and regulating protein synthesis/degradation

doi: 10.3389/fphar.2022.1056460

Figure Lengend Snippet: Proposed schematic diagram illustrating the protective efficacy, mechanisms and safety of morroniside against denervation-induced skeletal muscle atrophy. TNFα is elevated in muscle induced by denervation, activating NF-κB signaling, which is associated with atrophying muscle mass in mice and grip strength in population identified by genetic association studies. Morroniside attenuates TNFα-induced C2C12 myotubes atrophy via inhibiting canonical and non-canonical NF-κB signaling, inflammatory mediators and zinc accumulation pathway, proteasomal and autophagic protein degradation pathways, while improving protein synthesis pathway. In denervation-induced mice, morroniside decreases TNFα levels, canonical and non-canonical NF-κB signaling, proteasomal degradation pathway and increases weight of body, TA, EDL, Sol, Gas muscles, as well as Pax7+ satellite cells, cross section area of MyHC IIA, IIB, IIX myofibers and the percentage of MyHC IIA myofibers without obvious liver and kidney toxicity, therefore ameliorating muscle atrophy.

Article Snippet: For primary myogenic cells and C2C12 cells differentiated myotube staining, primary antibody to MyHC (1:100, R&D Systems, Minneapolis, MN, United States, catalog MAB4470) was incubated at 4°C overnight.

Techniques: Muscles

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Excessive DAO inhibits myoblast migration, leading to impaired myotube fusion and muscle strength decline by reducing ECM

doi: 10.3389/fbioe.2025.1606357

Figure Lengend Snippet: DAO content and muscle strength and muscle fibre types in natural aged mice and rapidly aged mice. (A) Grip strength was measured in 4-month-old (young) and 18-month-old (old) male C57 mice using a dynamometer for 4 days. The older group showed significantly reduced grip strength (** p < 0.01). (B,C) Western blot analysis of gastrocnemius (GA) muscle lysates showed increased expression of aging markers p53 and p16, along with elevated DAO content in the older group, three replicates were showed in each group. (D,E) Immunohistochemistry of GA and tibialis anterior (TA) muscles confirmed higher DAO content (brown) in the older group. Scale bars: 200 μM. Three different area were randomly selected to measure the positive area (%). (F) Immunofluorescence staining of tibialis anterior (TA) and gastrocnemius (GA) muscles in 6-month-old male SAMR1 (control) and SAMP8 (accelerated aging) mice. DAO (red) was co-stained with MYH7 (slow muscle fiber marker, green) and MYH1 (fast muscle fiber marker, blue). The merge images show the distribution of muscle fibers. Scale bars: 200 μM.

Article Snippet: Immunofluorescence: Sections were stained with antibodies against MYH1 (Anti-Fast Myosin Skeletal Heavy chain Rabbit pAb, GB112130 , Servicebio, Wuhan, China) and MYH7 (Anti-Slow Skeletal Myosin Heavy chain Mouse mAb, GB121857 , Servicebio, Wuhan, China), followed by appropriate secondary antibodies.

Techniques: Western Blot, Expressing, Immunohistochemistry, Muscles, Immunofluorescence, Staining, Control, Marker