Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (9): 1904-1912.doi: 10.3864/j.issn.0578-1752.2020.09.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Effects of GnIH on Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells via p38MAPK Signaling Pathway

Xin ZHANG,KongLin HUO,XingXing SONG,DuoNi ZHANG,Wen HU,ChuanHuo HU(),Xun LI()   

  1. College of Animal Science and Technology, Guangxi University, Nanning 530004
  • Received:2018-11-30 Accepted:2020-03-11 Online:2020-05-01 Published:2020-05-13
  • Contact: ChuanHuo HU,Xun LI E-mail:hch64815@gxu.edu.cn;lixun198@163.com

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Abstract:

【Objective】Studies have shown that autophagy and apoptosis restrict each other. As one of the main regulatory pathways of apoptosis, p38MAPK signaling pathway also has the dual effects of promoting and inhibiting autophagy. It has been proved that gonadotropin inhibitory hormone (gonadotropin-inhibitory hormone, GnIH) has effects on autophagy and apoptosis, but the mechanism of action is not clear. This experiment was conducted to study the effects of GnIH on autophagy and apoptosis of porcine ovarian granulosa cells via p38MAPK signaling pathway and its mechanism. 【Method】Oval granulosa cells were extracted from pig ovaries and cultured in vitro. To explore the best time of GnIH on p38MAPK signaling pathway, according to the time gradient of incubation GnIH (0, 10, 30, 60, and 90 min), Western blot was used to detect the protein expression of p38 and p-p38 in pGCs. To verify the effect of GnIH on p38MAPK signaling pathway, the cells were divided into 4 groups (control, GnIH, p38 activating agent (U-46619), and U-46619 +GnIH), Western blot was used to detect the protein expression of p38 and p-p38. To investigate the effects of different concentrations of GnIH on autophagy and apoptosis: the cells were divided into 5 groups ( control, 10 -6mol·L -1 GnIH, 10 -8mol·L -1 GnIH, 10 -10mol·L -1 GnIH, and 10 -12mol·L -1 GnIH), Western blot was used to detect the protein expression of autophagy and apoptosis. To verify the effects of different concentrations of GnIH on autophagy and apoptosis through p38 signaling pathway: the cells were divided into 6 groups (control, U-46619, U-46619+10 -6 mol·L -1 GnIH, U-46619+10 -8mol·L -1 GnIH, U-46619+10 -10mol·L -1 GnIH, and U-46619+10 -12mol·L -1 GnIH), Western blot was used to detect the protein expression of autophagy and apoptosis. 【Result】After incubation with GnIH for 10 min, the protein expression of p38 and p-p38 was significantly decreased (P<0.05). The results suggested that the optimum action time of GnIH on p38MAPK signaling pathway was 10 min; U-46619 significantly promoted the phosphorylation of p38 in pGCs (P<0.05), while GnIH significantly inhibited p38 phosphorylation of pGCs (P<0.05). The results suggested that U-46619 activated the p38MAPK signaling pathway, and GnIH inhibited the activation of p38MAPK signaling pathway; When the concentration of GnIH was 10 -6 mol·L -1, the autophagy and apoptosis of pGCs increased significantly (P<0.05). With the decrease of GnIH concentration, the autophagy level of pGCs increased gradually (P<0.05), while the apoptosis level of pGCs decreased gradually (P<0.05). The results suggest that high concentration of GnIH promote autophagy and apoptosis. With the decrease of GnIH concentration, the autophagy level increased gradually, while the apoptosis level decreased gradually. After adding U-46619, GnIH significantly upregulated the autophagy of pGCs and down-regulated the apoptosis of pGCs (P<0.05), which suggested that different concentrations of GnIH affected the autophagy and apoptosis of pGCs through p38MAPK signaling pathway. 【Conclusion】GnIH might up-regulate the autophagy of pGCs and reduce the apoptosis of pGCs by inhibiting the activation of p38MAPK signaling pathway.

Key words: autophagy, apoptosis, GnIH, p38MAPK, porcine, ovarian granulosa cells

Fig. 1

Immunoblot and statistical analysis of p38 and p-p38 proteins with different time of RFRP-3 (0, 10, 30, 60, 90 min, n=3) Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

Fig. 2

Immunoblot and statistical analysis of p38 and p-p38 proteins (control, GnIH, U-46619, GnIH+U-46619) (n=3) Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

Fig. 3

RFRP-3 concentration gradient (0, 10-6, 10-8, 10-10, 10-12) treated with pGCs.Western blot caspase-3, Bax and Bcl-2 and statistical analysis (n=3) Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

Fig. 4

RFRP-3 concentration gradient (0, 10-6, 10-8, 10-10, 10-12) treated with pGCs. Western blot Beclin-1, Atg12 and Atg5 and statistical analysis (n=3) Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E.M. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

Fig. 5

After incubated with p38 activator, RFRP-3 concentration gradient (0, 10-6, 10-8, 10-10, 10-12) was treated with pGCs.Western blot caspase-3,Bax and Bcl-2 and statistical analysis (n=3) Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

Fig. 6

After incubated with p38 activator, RFRP-3 concentration gradient (0, 10-6, 10-8, 10-10, 10-12) was treated with pGCs. Western blot Beclin-1,LC3 and Atg-5 and statistical analysis Densitometric quantification was performed using ImageJ with GAPDH as the internal control for normalization. Values are mean±S.E. Compared with control group, asterisk indicates significant difference. *P<0.05; **P<0.01"

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