Recombinant Human Endostatin Combined With Radiation Promotes Myocardial Fibrosis in Rat via TGF-β1/Smads/CTGF Signaling Pathway

Abstract

Recombinant human endostatin (rh-ES) has been found to cause heart damage in clinical use. The heart tissue can be irradiated inevitably while radiation therapy for chest tumors and may result in radiation damage. The regulation of myocardial fibrosis may be related to TGF-β1, Smad2/3, and CTGF signaling pathways. At present, whether targeted drug therapy combined with radiotherapy will increase the damage of the heart is a subject worth studying.The experimental animals were randomly divided into 25 Gy irradiation group (5 Gy/ time × 5 days), rh-ES 6mg/kg group, rh-ES 12mg/kg group, 25 Gy irradiation + rh-ES 6mg/kg group, 25 Gy irradiation + rh-ES 12mg/kg group and blank control group, with 15 in each group. rh-ES was injected intraperitoneally for 14 consecutive days. Five rats in each group were sacrificed and the cardiac tissues were extracted for HE staining, Masson staining and transmission electron microscopy at 1, 3 and 6 months after irradiation and rh-ES rejection. MTT was used to detect the value of the IC50 rh-ES to cardiomyocytes with different concentrations of rh-ES. The cardiomyocytes with stable knockdown of TGF-β1 is grouped, the irradiation dose is 10 Gy. According to cardiomyocytes trial were divided into four group, the blank control group (without any treatment), the irradiation group (irradiation + TGF-β1 gene knockdown), the rh-ES group (rh-ES + TGF-β1 gene knockdown), the combined group (irradiation, rh-ES and TGF-β1 gene knockdown), flow cytometry was used to detect cardiomyocytes apoptosis, qPCR and Western Blot were used to detect the expression of TGF-β1/Smad2. Smad3/CTGF gene and protein.In vivo experiments on the heart of rats showed that a little inflammatory cell infiltration was observed under light microscope at 1 month after radiation and rh-ES treatment, and the inflammatory cell infiltration was obvious with fibrosis changes at 3 months, and the fibrosis changes were aggravated 6 months later. rh-ES alone caused slight injury to rats (no change under light microscope, but some mitochondria swelling and cristae missing under electron microscope). rh-ES and radiation treatment increased the damage of myocardial tissues in rats to some extent, and the process of radiation-induced myocardial fibrosis lasted at least 6 months after radiotherapy. rh-ES can aggravate the process of myocardial fibrosis after radiation injury, but has no toxic effect on normal myocardial tissue. The IC50 value of rh-ES on rat cardiomyocytes was 529μg/ mL in vitro experiment. qPCR and Western Blot results showed that TGF-β1, Smad2/Smad3, CTGF gene and protein expressions were up-regulated in rat cardiomyocytes after rh-ES and irradiation treatment.The TGF-β1, Smad2/Smad3 and CTGF signaling pathways may be the common signaling pathways of myocardial fibrosis injury induced by rh-ES combined with radiation, which may be the target for the treatment of myocardial fibrosis by radiation.