Effect and Mechanism Analysis of Melittin on Promoting Muscle Differentiation of Adipose Stem Cells

Abstract

PurposeMelittin is the main peptide component of melittin venom, with anti-allergic, anti-inflammatory, anti-arthritis, anti-cancer, and neuroprotective effects. Melittin has been confirmed to have anti-inflammatory effects in immune and liver cells and is widely used in treating arthritis and rheumatic diseases. At the same time, related studies have shown that melittin can enhance the expression of muscle differentiation related factors in mice. However, whether the melittin can improve the expression of stem cells into muscle differentiation and use it to treat Skeletal muscle atrophy, it has not yet been clarified.As "seed cells," stem cells have been widely concerned in the field of regenerative medicine research, such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, etc., among which adipose mesenchymal stem cells (ADSCs) have excellent application potential in the treatment of Skeletal muscle atrophy because of their quantitative advantages, easy access, and inherent good differentiation potential and self-renewal ability. To investigate whether melittin can stimulate ADSCs MYOGenic differentiation and whether combined with ADSCs can treat Skeletal muscle atrophy. MethodsO_LIFour concentration ranges of melittin were set, namely Group A (20 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L), Group B (2 mg/L, 4 mg/L, 8 mg/L, 16 mg/L, 32 mg/L), Group C (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L), and Group D (0.02 mg/L, 0.04 mg/L, 0.08 mg/L, 0.16 mg/L, 0.32 mg/L), Intervention of adipose stem cells with different concentrations of melittin for 48 hours, followed by measurement of relative cell viability using the CCK-8 method. C_LIO_LIMelittin after 48 hours of intervention with ADSCs, Western blot was used to detect the expression levels of MYOGenic differentiation related factors. C_LIO_LIThe control group (C), melittin group (M), dexamethasone group (D), and Dexamethasone+Melittin group (D+M) were set separately. After 12 hours of intervention with dexamethasone (10umol/L), the ADSCs were added with melittin for 48 hours. Western blot was used to detect MYOGenic differentiation related factors MYOGenic(MYOG), MYF5, MYOD, MYH4 p38 and p-p38 protein. In order to observe the process of melittin repairing cell atrophy, detected MYOD and MYOG using immunofluorescence. C_LIO_LIRandomly divided into control group (C), damage group (T), stem cell therapy group (SC), melittin combined with ADSCs therapy group (SC+M). Group C does not do any treatment. Group T, SC, and SC+M set up a mouse shoulder sleeve injury model, all from the left gang of the rats, and the suture is marked with surgical sutures. On the first day of surgery, The SC group of the muscle injecting ADSCs(1x106)in the SC group, and the ADSCs after the melittin intervention of the SC+M mouse is repeatedly treated every seven days. On the left, the tendon is extracted, and the muscles are extracted to perform related tissue-related testing such as Su Mu Jing-Yi Hong dyed (H&E dyed), immunohistochemistry, muscle fiber weight and diameter. C_LI ResultsO_LICCK-8 Experiment shows that the suitable concentration of Melittin has no significant toxic effect on ADSCs. C_LIO_LIWestern blot experiment shows that the appropriate concentration of Melittin can enhance the expression of muscle differentiation related factors in ADSCs. C_LIO_LIWestern blot and immunofluorescence staining results show that the expression related factors of the D+M group are better than that of group D, and the expression related factors of forming muscle differentiation in group M is also better than group C. C_LIO_LIMelittin can promote ADSCs muscle differentiation through the p38-MAPK signaling pathway. C_LIO_LIAnimal experiments have shown that the expression related factors of the SC+M treatment groups muscle differentiation related factors is significantly higher than that of the SC therapy group, and the expression related factors of the two groups of rats of SC+M and SC groups are better than the T group Rat. Organic research has confirmed that rats who have been treated with ADSCs treated with melittin have significantly improved in muscle weight, muscle fiber diameter, and muscle fiber cross-sectional area. At the same time, the result of immunohistochemicals shows that the expression of MYOD and MYOG in the SC+M group is better than the SC group. C_LI ConclusionThe results of this study indicate that melittin can promote MYOGenic differentiation of ADSCs and increase the expression of MYOGenic differentiation related factors in atrophic ADSCs.Secondly, melittin can activate the p38-MAPK signaling pathway and promote the MYOGenic differentiation of ADSCs. Stem cells treated with melittin intervention can improve the recovery of muscle mass, muscle fiber diameter, and muscle fiber cross-sectional area in rats with skeletal muscle injury compared to rats treated with stem cells alone. Therefore, the above results provide insights into MYOGenic differentiation and may provide a potential alternative strategy for related diseases such as skeletal muscle atrophy.