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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (3): 400-411. doi: 10.19723/j.issn.1671-167X.2022.03.003

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Curcumin alleviates the manganese-induced neurotoxicity by promoting autophagy in rat models of manganism

Li-ye LAI,Chang-song DOU,Cui-na ZHI,Jie CHEN,Xue MA,Peng ZHAO,Bi-yun YAO*()   

  1. Department of Toxicology, Peking University School of Public Health, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China
  • Received:2022-02-12 Online:2022-06-18 Published:2022-06-14
  • Contact: Bi-yun YAO E-mail:yaoby@bjmu.edu.cn
  • Supported by:
    Beijing Natural Science Foundation(7192099);Beijing Natural Science Foundation(7150284)

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Abstract:

Objective: To investigate the protective effects of curcumin(CUR) and its mechanism on a rat model of neurotoxicity induced by manganese chloride (MnCl2), which mimics mangnism. Methods: Sixty male SD rats were randomly divided into 5 groups, with 12 rats in each group. Control group received 0.9% saline solution intraperitoneally (ip) plus double distilled water (dd) H2O intragastrically (ig), MnCl2 group received 15 mg/kg MnCl2(Mn2+ 6.48 mg/kg) intraperitoneally plus dd H2O intragastrically, CUR group received 0.9% saline solution intraperitoneally plus 300 mg/kg CUR intragastrically, MnCl2+ CUR1 group received 15 mg/kg MnCl2 intraperitoneally plus 100 mg/kg curcumin intragastrically, MnCl2+ CUR2 group received 15 mg/kg MnCl2 intraperitoneally plus 300 mg/kg CUR intragastrically, 5 days/week, 4 weeks. Open-field and rotarod tests were used to detect animals' exploratory behavior, anxiety, depression, movement and balance ability. Morris water maze (MWM) experiment was used to detect animals' learning and memory ability. ICP-MS was used to investigate the Mn contents in striata. The rats per group were perfused in situ, their brains striata were removed by brains model and fixed for transmission electron microscope (TEM), histopathological and immunohistochemistry (ICH) analyses. The other 6 rats per group were sacrificed. Their brains striata were removed and protein expression levels of transcription factor EB (TFEB), mammalian target of rapamycin (mTOR), p-mTOR, Beclin, P62, microtubule-associated protein light chain-3 (LC3) were detected by Western blotting. Terminal deoxynucleotidyl transterase-mediated dUTP nick end labeling (TUNEL) staining was used to determine neurocyte apoptosis of rat striatum. Results: After exposure to MnCl2 for four weeks, MnCl2-treated rats showed depressive-like behavior in open-field test, the impairments of movement coordination and balance in rotarod test and the diminishment of spatial learning and memory in MWM (P < 0.05). The striatal TH+ neurocyte significantly decreased, eosinophilic cells, aggregative α-Syn level and TUNEL-positive neurocyte significantly increased in the striatum of MnCl2 group compared with control group (P < 0.05). Chromatin condensation, mitochondria tumefaction and autophagosomes were observed in rat striatal neurocytes of MnCl2 group by TEM. TFEB nuclear translocation and autophagy occurred in the striatum of MnCl2 group. Further, the depressive behavior, movement and balance ability, spatial learning and memory ability of MnCl2+ CUR2 group were significantly improved compared with MnCl2 group (P < 0.05). TH+ neurocyte significantly increased, the eosinophilic cells, aggregative α-Syn level significantly decreased in the striatum of MnCl2+ CUR2 group compared with MnCl2 group. Further, compared with MnCl2 group, chromatin condensation, mitochondria tumefaction was alleviated and autophagosomes increased, TFEB-nuclear translocation, autophagy was enhanced and TUNEL-positive neurocyte reduced significantly in the striatum of MnCl2+ CUR2 group (P < 0.05). Conclusion: Curcumin alleviated the MnCl2-induced neurotoxicity and α-Syn aggregation probably by promoting TFEB nuclear translocation and enhancing autophagy.

Key words: Manganism, Curcumin, Transcription factor EB (TFEB), Autophagy

CLC Number: 

  • R135.1

Table 1

The effects of CUR supplementation and MnCl2 administration on rat body weights  /g"

Group Week 1 Week 2 Week 3 Week 4
NC 216.92±12.6 281.38±12.30 347.31±17.74 406.46±21.64
CUR 219.17±9.10 286.61±11.88 354.83±25.39 397.87±25.37
MnCl2 214.38±12.5 242.94±17.15* 306.70±22.58* 353.00±25.16☆
MnCl2+ CUR1 222.89±7.40 251.59±21.95 324.60±21.85 365.54±32.04*
MnCl2+ CUR2 221.74±9.40 259.04±16.30 323.93±16.30 369.27±22.27

Table 2

The effects of CUR supplementations and MnCl2 administration on Mn content in striatum of rats"

Group Mn2+ content/(ng/g)
NC 430.06±41.63
CUR 434.32±45.87
MnCl2 842.39±68.05☆
MnCl2+ CUR1 781.99±82.86☆
MnCl2+ CUR2 817.24±49.14☆

Table 3

The effects of CUR supplementation and MnCl2administration on neurobehavioral performance of male rats by open field test and rotarod test"

Test Indicators NC CUR MnCl2 MnCl2+CUR1 MnCl2+CUR2
OFT Numbers of rearing 18.25±5.37 17.63±5.85 10.63±2.45☆ 12.00±4.43* 17.37±4.98★
Numbers of entries to the inner zone 5.38±3.42 6.63±3.25 4.00±2.83* 4.83±1.83 5.13±2.64#
Speed in inner zone/ (m/s) 0.18±0.07 0.19±0.05 0.23±0.09 0.18±0.07 0.17±0.03
Distance moved in the inner zone/m 2.32±0.59 2.22±0.49 1.17±0.25* 1.44±0.30 2.25±0.38#
Time in the inner zone/s 13.16±2.40 13.38±3.04 5.69±1.22* 8.02±1.49* 13.26±2.10#
Rotarod Time on rotarod/s 98.86±19.5 99.41±17.98 61.98±23.04☆ 72.79±16.46* 88.70±19.65#
Circles on rotarod 17.21±5.07 16.80±4.68 8.35±4.10☆ 10.63±3.87* 12.73±5.02#
Fall speed/(m/s) 15.76±2.35 15.72±2.09 11.22±2.58 12.62±1.99 13.80±2.34

Figure 1

The effects of CUR supplementation and MnCl2 administration on spatial learning ability of male rats by navigation test of MWM NC, negative control; CUR, curcumin. $\bar x \pm s$, n=12，*P < 0.05, ☆P < 0.01, compared with NC group; ★P < 0.01，compared with MnCl2 group."

Table 4

The effects of CUR supplementation and MnCl2 administration on the memory performance by explore test of Morris water maze"

Items NC CUR MnCl2 MnCl2+ CUR1 MnCl2+ CUR2
Number of entries to the island 5.13±0.64 4.33±1.60 3.38±1.30* 4.83±1.33 5.13±0.84#
Number of entries to the target quadrant 10.13±0.84 9.75±1.03 9.13±1.81 10.67±1.21 10.50±2.51
Time in the target quadrant/s 41.24±5.56 42.50±5.59 34.75±6.17* 33.98±2.92* 42.84±6.65#
Distance in the target quadrant/m 9.06±0.91 9.31±1.62 7.63±1.44* 8.69±0.52 9.54±1.42#
Target quadrant time/% 35.50±5.0 35.70±4.6 29.30±5.1☆ 28.20±2.5☆ 35.80±5.6★
Target quadrant distance/% 29.10±2.1 30.40±3.5 26.30±4.4* 27.40±2.4 29.40±2.7#
Average speed/(m/s) 0.25±0.01 0.22±0.02 0.24±0.04 0.27±0.03 0.26±0.06
Total distance travelled/m 29.79±1.70 30.60±2.79 29.10±2.95 32.51±3.98 32.51±4.95

Figure 2

The effects of CUR supplementation on MnCl2-induced injury of striatal neurocyte of male rats A, DAB staining of TH by ICH(scale bars=1 000 μm)；B, the quantitative results of A; C, the HE staining of coronal section of the striatum (scale bars = 200 μm，the red arrows stood for eosinophilic cells); D, the quantitative results of eosinophilic cells of C. a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. $\bar x \pm s$，n=6，*P < 0.05, ☆P < 0.01, compared with NC group; #P < 0.05, ★ P < 0.01，compared with MnCl2 group."

Figure 3

The effects of CUR supplementation on the MnCl2-induced ultrastructures alteration of striatal neurocytes of male rats a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. Nucleus, yellow arrows, mitochondria, blue arrows; autophagosome, red arrows."

Figure 4

The effects of CUR supplementation on the MnCl2-induced alteration in α-Syn expression in striatum of male rats A, DAB staining of α-Syn by ICH (scale bars = 200 μm); B, the brand of α-Syn by Western blotting；C, the semi-quantitive analysis of density of the protein bands normalized to that of β-actin of B. a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. $\bar x \pm s$, n=6, *P < 0.05, ☆P < 0.01, compared with NC group; ★P < 0.01, compared with MnCl2 group."

Figure 5

The effects of CUR supplementation on the MnCl2-induced alteration in the cellular localization and expression of TFEB in striatum A, the brand of TFEB by Western blotting；B, the semi-quantitive analysis of density of the protein bands normalized to that of GAPDH/H3 of A. a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. $\bar x \pm s$，n=6. *P < 0.05, ☆P < 0.01, compared with NC group; # P < 0.05, ★P < 0.01, compared with MnCl2 group."

Figure 6

The effects of CUR supplementation on the MnCl2-induced alteration in mTOR, p-mTOR, Beclin1, P62 and LC3 expression in striatum A, the brands of autophagy-related protein by Western blotting；B, the semi-quantitive analysis of density of the protein bands normalized to that of β-actin of A. a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. $\bar x \pm s$，n=6. *P < 0.05, ☆P < 0.01, compared with NC group; # P < 0.05, ★P < 0.01, compared with MnCl2 group."

Figure 7

The effects of CUR supplementations on the MnCl2-induced apoptosis of striatal neurocyte of male rats A, TUNEL staining of rats’ striatum (scale bars = 200 μm); B, the number of TUNEL-positive cells of rats’ striatum. a, negative control (NC); b, curcumin (CUR); c, MnCl2; d, MnCl2+ CUR1; e, MnCl2+ CUR2. $\bar x \pm s$，n = 6，☆P < 0.01, compared with NC group; ★P < 0.01, compared with MnCl2 group."

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