Prokaryotic expression, purification and evaluation of anti-cardiac fibrosis activity of recombinant TGF-β latency associated peptide
Authors: Song Xudong , Qiu Yufei , Shi Jiayi , Li Luxin , Yuan Xiaohuan , Wu Dan , Chu Yanhui 
Published: January 2022

Background
Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix in the heart, which leads to the formation of cardiac scars. It causes systolic and diastolic dysfunction, and ultimately leads to cardiac dysfunction and arrhythmia. TGF-β1 is an important regulatory factor involved in cardiac fibrosis. Studies have shown that the N-terminal latency associated peptide (LAP) must be removed before TGF-β1 is activated. We hypothesize that recombinant LAP may inhibit cardiac fibrosis induced by TGF-β1. To evaluate anti-cardiac fibrosis activity of recombinant LAP, an experimental study was carried out and is reported here.

Methods
The pET28a-LAP plasmid was constructed and transformed into E. coli C43 (DE3) competent cells. The recombinant LAP protein was purified by Ni affinity chromatography. The cells were treated with TGF-β1 at different concentrations for 24 h. The expression of α-SMA was detected by Western blot. RTCA was used to detect the effect of recombinant LAP on the proliferation of H9C2 cells induced by 10 ng/mL TGF-β1. To detect the effect of LAP on the expression of fibrosis-related proteins, H9C2 cells were treated with 10 ng/mL TGF-β1 for 24 h, then added 60 μg/mL recombinant LAP for 48 h. The LAP group was treated with 60 μg/mL recombinant LAP alone. The LAP pre-protection group was treated with 10 ng/mL TGF-β1 and 60 μg/mL recombinant LAP at the same time. Western blot and immunofluorescence were used to detect the expression of α-SMA, collagen I and fibronectin and p-Smad2.

Results
The recombinant LAP was prokaryotic expressed and purified. 10 ng/mL was determined as the optimal working concentration of TGF-β1 to induce H9C2 cells fibrosis. RTCA results showed that 60 μg/mL LAP could effectively inhibit the proliferation of H9C2 cells induced by TGF-β1. Immunofluorescence results showed that compared with the control group, the fluorescence intensities of α-SMA, collagen I and FN increased significantly after TGF-β1 treatment. The fluorescence intensities in the TGF-β1+LAP group decreased significantly. Western blot results showed that 60 μg/mL LAP could inhibit the increase of α-SMA, collagen I and FN expression in H9C2 cells induced by TGF-β1. Compared with the control, the LAP alone group has no significant difference in α-SMA and p-Smad2 expression level. The expression of α-SMA and p-Smad2 in the TGF-β1 model group was significantly increased compared with the control group. Compared with the TGF-β1 group, both TGF-β1+LAP group and LAP pre-protection group significantly reduced the increase in α-SMA and p-Smad2 levels.

Conclusions
Recombinant LAP was prokaryotic expressed and purified. The results showed that recombinant LAP can inhibit the cell proliferation and expression increase of α-SMA, collagen I, fibronectin and p-Smad2 in H9C2 cells induced by TGF-β1. These results suggested that recombinant LAP might inhibit TGF-β1-induced fibrosis of H9C2 cells through the TGF-β/Smad pathway.