SIRT3 Inhibits Mitophagy and Alleviates Neuronal Oxygen Deprivation/Reoxygenation Injury Aggravated by High Glucose

doi:10.13523/j.cb.2106024

China Biotechnology

2021, Vol. 41

Issue (11): 1-13 DOI: 10.13523/j.cb.2106024

SIRT3 Inhibits Mitophagy and Alleviates Neuronal Oxygen Deprivation/Reoxygenation Injury Aggravated by High Glucose

Chen-yang ZHANG¹,Chang-chun HEI²,Shi-lin YUAN¹,Yu-jia ZHOU¹,Mei-ling CAO¹,Yi-xin QIN¹,Xiao YANG^3,^**(

)

1 School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
2 Department of Human Anatomy, Histology and Embryology, Ningxia Medical University, Yinchuan 750004, China
3 Neuroscience Center, General Hospital of Ningxia Medical University, Yinchuan 750004, China

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Abstract

Objective: To investigate whether high glucose can aggravate oxygen deprivation/reoxygenation (OD/R) injury in neurons through regulating mitophagy and the specific molecular mechanism of regulating mitophagy. Methods: HT22 cells are used to establish a model of cellular oxygen deprivation/reoxygenation to simulate cerebral ischemia/reperfusion injury in vivo. And cells are treated with high glucose medium (HG 50 mmol/L). 3-TYP is given to inhibit the expression of SIRT3. CCK8 is used to examine cell viability; flow cytometry is used to detect cell apoptosis, and TMRE fluorescence detection kit is used to detect cell MMP; Western blot is used to detect the expression of mitochondrial division/fusion related protein (DRP1, OPA1, TOM20), autophagy related protein (LC3Ⅱ, Beclin-1), mitophagy related protein (PINK1, Parkin) and SIRT3. Results: Compared with OD groups, the cell density is further reduced, the cell contour is more blurred, the synapses are shortened, the floating cells increase in HG+OD groups, and cells in HG+OD groups have lower survival rate and higher apoptosis rate (P<0.05). Compared with OD groups, cells in HG+OD groups have lower MMP (P<0.05). Compared with OD groups, the expression of P-DRP1 in HG+OD groups is increased, but OPA1 and TOM20 are decreased (P<0.05); the expression of Beclin-1 and LC3Ⅱ as well as PINK1 and Parkin in HG+OD groups are also increased (P<0.05). Furthermore, SIRT3 protein and gene are further increased in HG+OD groups (P<0.05). 3-TYP treatment can further aggravate the OD/R injury of neurons in HG groups. And 3-TYP also increases the expression of DRP1, LC3Ⅱ and PINK1. Conclusions: High glucose can aggravate oxygen deprivation/reoxygenation injury in neurons by exacerbation of cell apoptosis, reduction of MMP and activation of mitophagy. In addition, SIRT3 can inhibit PINK1-Parkin pathway-mediated mitophagy and alleviate high glucose aggravated-neuronal OD/R injury.

Key words： Oxygen deprivation/reoxygenation High glucose Mitophagy Mitochondrial fusion and fission SIRT3

Received: 15 June 2021 Published: 01 December 2021

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Corresponding Authors: Xiao YANG E-mail: cckk606@sina.com

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	Chen-yang ZHANG
	Chang-chun HEI
	Shi-lin YUAN
	Yu-jia ZHOU
	Mei-ling CAO
	Yi-xin QIN
	Xiao YANG

Cite this article:

Chen-yang ZHANG,Chang-chun HEI,Shi-lin YUAN,Yu-jia ZHOU,Mei-ling CAO,Yi-xin QIN,Xiao YANG. SIRT3 Inhibits Mitophagy and Alleviates Neuronal Oxygen Deprivation/Reoxygenation Injury Aggravated by High Glucose. China Biotechnology, 2021, 41(11): 1-13.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106024 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I11/1

Table 1 The concentration of chemical substance in the solution of the model (单位:mmol/L)

Fig.1 Experimental protocol and experimental timeline for HG and OD/R treatment

Table 2 Primer sequences

Fig.2 The effect of high glucose on cells death in neurons after OD/R (a) Cell morphology was observed under microscope (b) Cell survival rate was detected by CCK8 (c),(d) Cell apoptotic rate was detected by flow cytometry Mean±SD, n=3; &&&. P<0.001 vs NC; **.P<0.01, ***.P<0.001 vs non-OD under the same condition; #. P<0.05, ###. P<0.001. Bar=200 μm

Fig.3 The effect of high glucose on mitochondrial membrane potential in neurons after OD/R (a),(b) Mitochondrial membrane potential was observed using a TMRE probe Mean±SD,n=3; &&&.P<0.001 vs NC; **.P<0.01, ***.P<0.001 vs non-OD under the same condition; #.P<0.05, ###.P<0.001. Bar=100 μm

Fig.4 The effect of high glucose on mitochondrial dynamic balance in neurons after OD/R (a),(b) P-DRP1 expression was monitored by Western blot (a),(c) OPA1 expression was monitored by Western blot (a),(d) TOM20 expression was monitored by Western blot Mean±SD, n=3;&&. P<0.01, &&&. P<0.001 vs NC; *. P<0.05, **. P<0.01, ***,P<0.001 vs non-OD under the same condition; #.P<0.05, ##, P<0.01, ###.P<0.001

Fig.5 The effect of high glucose on mitophagy in neurons after OD/R (a),(b) LC3Ⅱ expression was monitored by Western blot (a),(c) Beclin-1 expression was monitored by Western blot (d),(f) PINK1 expression was monitored by Western blot €,(g) Parkin expression was monitored by Western blot Mean±SD, n=3;&. P<0.05, &&. P<0.01, &&&. P<0.001 vs NC; *.P<0.05, **. P<0.01, ***. P<0.001 vs non-OD under the same condition; #. P<0.05, ##. P<0.01

Fig.6 The effect of high glucose on SIRT3 in neurons after OD/R (a) The gene expression of SIRT3 was detected by PCR (b),(c) The protein expression of SIRT3 was monitored by Western blot Mean±SD,n=3; &&. P<0.01, &&&. P<0.001 vs NC; *.P<0.05, ***.P<0.001 vs non-OD under the same condition; #.P<0.05, ##. P<0.01

Fig.7 The effect of 3-TYP on SIRT3 in neurons after HG and OD/R treatment (a),(b) SIRT3 expression was monitored by Western blot Mean±SD,n=3;*.P<0.05 vs control under the same condition; ##. P<0.01 NG vs HG; △.P<0.05, △△. P<0.01 HG vs HG+3-TYP

Fig.8 The effect of the inhibition of SIRT3 on cells death in neurons after HG and OD/R treatment (a) Cell survival rate was detected by CCK8 (b),(c) Cell apoptotic rate was detected by flow cytometry Mean±SD,n=3;*.P<0.05, **. P<0.01, ***.P<0.001 vs Control under the same condition; #.P<0.05, ##. P<0.01, ###.P<0.001 NG vs HG; △.P<0.05, △△. P<0.01 HG vs HG+3-TYP

Fig.9 The effect of the inhibition of SIRT3 on mitophagy in neurons after HG and OD/R treatment (a),(d) DRP1 expression was monitored by Western blot (b),(e) LC3Ⅱ expression was monitored by Western blot (c),(f) PINK1 expression was monitored by Western blot Mean±SD, n=3; **. P<0.01, ***.P<0.001 vs Control under the same condition; #.P<0.05, ##. P<0.01 NG vs HG; △.P<0.05, △△. P<0.01, △△△.P<0.001 HG vs HG+3-TYP


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