Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (9): 1921-1930.doi: 10.3864/j.issn.0578-1752.2020.09.018

• RESEARCH NOTES • Previous Articles    

Effect of Recombinant GSTA3 Protein on Expression of the Anti-Apoptotic Gene BAG-3 in Thiram-Induced Tibial Chondrodysplasia

Zhen LI,ShiXiong YANG,Sheng NIU,Ning ZHANG,Xin LI,YangYang ZHANG,YunFei JIA,ZhiXiong TIAN,GuanBao NING,Ding ZHANG,WenXia TIAN()   

  1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2019-05-20 Accepted:2019-12-26 Online:2020-05-01 Published:2020-05-13
  • Contact: WenXia TIAN E-mail:wenxiatian@126.com

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Abstract:

【Objective】Tibial chondrodysplasia (TD) is a common skeletal disease in broilers. The study was conducted to investigate the effect of recombinant GSTA3 protein on the expression of anti-apoptotic gene BAG-3 in the chondrocytes of broilers induced by thiram, so as to provide a new idea and method for the treatment of TD. 【Method】120 one-week-old broiler chicks were randomly divided into six groups (A, B, C, D, E and F), in which Group A, B and C were basic diet groups, and group D, E and F were thiram-containing diet groups. TD was induced by thiram for 2 days. After thiram was added for days 1, 3, 5 and 7, recombinant chicken GSTA3 protein and phosphate buffer were injected into the leg muscles. Group A and group D were injected with PBS (100 μg·kg -1); group B and group E were injected with low dose (100 μg·kg -1) GSTA3; group C and group F were injected with high dose (200 μg·kg -1) GSTA3. The experiment lasted 23 days. Tibia growth plates were collected at the 1st, 2nd, 4th, 6th, 10th and 15th days after thiram treatment. The mRNA level of BAG-3 gene was detected by quantitative PCR, and the protein expression of BAG-3 gene was observed by immunohistochemistry. 【Result】Real-time PCR results showed that the expression level of BAG-3 mRNA in tibia growth plate of broilers in the thiram-containing diet group was significantly increased, compared with that in the basal diet control group during the repair period of TD injury (P<0.05). Compared with the thiram-containing diet group, E and F groups had significant differences on days 2, 4, 10 and 15, and were significantly lower than that under the thiram-containing diet group on days 10 and 15 (P<0.05), indicating a faster recovery compared with group D. Immunohistochemical results showed that BAG-3 protein was not expressed in the proliferation area and the pre-hypertrophic area of the chicken tibial chondrocytes, but only in the cytoplasm of the hypertrophic area. Compared with the control group A, the expression of BAG-3 protein in the thiram-containing diet group was increased. The recombinant GSTA3 induced the protein expression level in the hypertrophic area in the high and low dose of GSTA3 group compared with the thiram-containing diet group D (days 10 and 15). 【Conclusion】In conclusion, GSTA3 recombinant protein could regulate the expression of BAG-3 to participate in the apoptosis pathway and inhibit the apoptosis during the induction of TD in broilers. During the TD-injury repair period, after GSTA3 injection, the expression of anti-apoptotic gene BAG-3 protein was enhanced, which could participate in cell apoptosis to alleviate the TD-injury and make the TD growth plate function of broilers return to normal quickly.

Key words: glutathione S-transferase A3 (GSTA3), tibial dyschondroplasia, BAG-3, antiapoptotic, broiler

Table 1

Test grouping"

分组Group 日粮Daily diet 注射物Injectant
A 基础日粮Basic diet PBS
B 基础日粮Basic diet 100 μg·kg-1重组GSTA3蛋白100 μg·kg-1 recombinant GSTA3 protein
C 基础日粮Basic diet 200 μg·kg-1重组GSTA3蛋白200 μg·kg-1 recombinant GSTA3 protein
D 福美双日粮Thiram diet PBS
E 福美双日粮Thiram diet 100 μg·kg-1重组GSTA3蛋白100 μg·kg-1 recombinant GSTA3 protein
F 福美双日粮Thiram diet 200 μg·kg-1重组GSTA3蛋白200 μg·kg-1 recombinant GSTA3 protein

Table 2

Primer design for Real-time PCR"

基因Gene 引物序列（5′→3′）Primer Sequence（5′→3′） 片段Size (bp) 退火温度Annealing temperature (℃)
BAG-3 F: TTCTGATTCTCCAACGGTTT
R: TTCATGGATCACAGGAATTG		 100 55
18S rRNA F：TTCCGATAACGAACGACAC
R：GACATCTAAGGGCATCACAG		 139 55

Fig. 1

Expression changes of BAG-3 in tibial growth plate on day 1 P: Proliferative zone; PH: Prehypertrophic zone; H: Hypertrophic zone. The same as below"

Fig. 2

Expression changes of BAG-3 in tibial growth plate on day 2"

Fig. 3

Expression changes of BAG-3 in tibial growth plate on day 4"

Fig. 4

Expression changes of BAG-3 in tibial growth plate on day 6"

Fig. 5

Expression changes of BAG-3 in tibial growth plate on day 10"

Fig. 6

Expression changes of BAG-3 in tibial growth plate on day 15"

Fig. 7

Expression of BAG-3 mRNA in growth plate of the TD broiler injected with different dosage of chGSTA3 protein A. Basic diet + PBS; B. Basic diet +100 μg·kg-1 recombinant GSTA3 protein; C. Basic diet +200 μg·kg-1 recombinant GSTA3 protein; D. Diet containing thiram + PBS injection; E. Diet containing thiram +100 μg·kg-1 recombinant GSTA3 protein; F. Diet containing thiram +200 μg·kg-1 recombinant GSTA3 protein; Different small letters indicate significant statistics difference(P<0.05)"

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