Safe and effective subcutaneous adipolysis in minipigs by a collagenase derivative.

Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.

Proliferative and anti-proliferative effects of thymosin alpha1 on cells are associated with manipulation of cellular ROS levels.

Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system and play important roles in a variety of physiological and pathological processes. Previous studies indicate that modulation of cellular ROS affects cell proliferation. Thymosin alpha 1 (Talpha1) is a naturally occurring thymic peptide and has previously been shown to be a potential therapy for some immunodeficiencies, malignancies, and infections. However, few reports have focused on manipulation of cellular ROS level effects of Talpha1. In this study, the Talpha1-treated leukomonocytes, which were isolated from mice spleens, exhibited a higher ROS level and a lower reduced glutathione (GSH) level; however, HepG2 cells treated with Talpha1 exhibited lower ROS level and higher GSH level. In addition, after treatment with Talpha1, the population of leukomonocytes in the G(2) phase increased, resulting in a slight increase in viability. However, in Talpha1-treated HepG2 cells, the cell cycle was delayed in the G(1) phase, thereby inhibiting tumor cell proliferation; in addition, dephosphorylation of the serine/threonine kinase Akt was detected. In conclusion, we show that Talpha1 has potent anti-proliferative activity against malignant human hepatoma cells and proliferative activity against leukomonocytes associated with manipulation of oxidative stress levels which indicates the potential of Talpha1 as an antitumor drug.