Nrf2 mediated ER-phagy protects against oxidative damage in intervertebral disc degeneration

Intervertebral disc degeneration (IDD) increases the risk of low back pain (LBP). Oxidative stress may induce cellular damage and contribute to various diseases including IDD. Endoplasmic reticulum autophagy (ER-phagy) is a specific type of autophagy, its role in oxidative stress induced damage as well as in IDD is unknown. This study explores the role of ER-phagy in oxidative damage in intervertebral disc nucleus pulposus cells (NPCs), as well as the Nrf2/FAM134B axis in ER-phagy regulation and IDD therapy. We found ER-phagy was decreased in NPCs during oxidative stress; while FAM134B may promote ER-phagy and alleviate oxidative stress induced ER-stress and apoptosis. In addition, the nuclear transcription factor Nrf2 may promote the expression of FAM134B as well as ER-phagy, and suppress ER-stress and apoptosis in NPCs. Furthermore, overexpression of FAM134B and Nrf2 could effectively attenuate the progression of IDD in rats in vivo. These results suggest Nrf2/FAM134B mediated ER-phagy may combat oxidative damage in cells; meanwhile, ER-phagy as well as Nrf2 could be potential therapeutic targets for IDD. Graphical abstract


Introduction
NPCs were lysed in ice-cold RIPA with 1 mM PMSF (phenylmethanesulfonyl 156 fluoride, Beyotime). The protein concentration in the samples was measured using the 157 BCA protein assay kit (Beyotime). The proteins were separated via sodium dodecyl 158 sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene 159 difluoride membrane (Millipore, USA) followed by blocking with 5% nonfat milk.

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The bands were subsequently probed with primary antibodies. Finally, the intensity of 161 the bands was quantified using Image Lab 3.0 software (Bio-Rad).    2.14. X-ray imaging acquisition 241 For X-ray imaging, it was performed for all rats at 0 week, 4 weeks, and 8 weeks after 242 surgery by the X-ray irradiation system (Kubtec, USA). After imaging, the height of 243 the intervertebral disc was measured using Image J software and the disc height index 244 (DHI) was calculated to reflect the changes in intervertebral disc height as described 245 in previous study.  Fig.1A). Using this reporter system, 275 it was found that the signal of GFP -mCherry + was high in control group, the treatment 276 of TBHP resulted in decreased signal of GFP -mCherry + (Fig. 1C), and the 277 quantification data showed that the ratio of GFP -mCherry + /GFP + mCherry + was dose 278 dependently decreased during TBHP treatment (Fig. 1C&D). 279 We then inhibit the activity of lysosome with the use of Bafilomycin A1 (Baf). Baf  (Fig. 1F). Furthermore, we observed autophagosomes and selective autophagy by 286 transmission electron microscopy (TEM). Compared to control group, TBHP-treated 287 group showed less the autophagosomes contained parts of ER whorls (some rough ER 288 parts with ribosomes adhesion) (Fig. 3G).

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In summary, these results suggest that ER-phagy occurs in NPCs, and is decreased 290 during oxidative stress. with LV-FAM134B, the autophagosomes contained more ER parts (Fig. 2I). Finally, 315 we used RAMP-GFP-mCherry and LV-FAM134B in NPCs. The process of ER-phagy 316 was captured by confocal microscope (Fig. 2J).

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In summary, it is suggested that the expression of FAM134B is decreased during 318 oxidative stress, while FAM134B can effectively regulate the level of ER-phagy in 319 NPCs.

FAM134B suppresses ER-stress and apoptosis in nucleus pulposus cells 322
Next, we sought to evaluate the function of ER-phagy on ER-stress and apoptosis. reduced the apoptosis of NPCs stimulated by oxidative stress (Fig. 3D&E). 337 Subsequently, we detect the effect of overexpression of FAM134B on apoptosis 338 through TUNEL staining, and we can get the same experimental conclusion as the 339 previous one (Fig. 3F). In addition, LAMP1 (488nm)-CANX (594nm)-C-C3 (647nm) is rescued (Fig. 3G).

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In summary, these results show that FAM134B medicate ER-phagy may suppresses 348 ER-stress and apoptosis in NPCs during oxidative stress.

ER-phagy 352
In the next step, we aimed to prove that FAM134B exerts its effects by regulating obvious lysosome accumulation was observed in the cells, suggesting that the 359 ER-phagy process was blocked (Fig. 4A). After that, we used western blot to verify 360 that the expression of apoptosis-related protein and ER-stress-related proteins. After 361 Baf treatment, the therapeutic effect of FAM134B overexpression was suppressed 362 (Fig.4B-G). Finally, we used CHOP (488nm) and BCL-2 (594nm) 363 immunofluorescence double staining and TUNEL staining to verify and draw similar 364 conclusions (Fig.4H&I). 365 Together, these results suggest that FAM134B exerts its effects by regulating 366 ER-phagy. 369 The results above show that ER-phagy may suppress oxidative damage in NPCs; 370 however, there is no specific agonist for ER-phagy so far. Thus, we aimed to explore 371 the upstream regulator of ER-phagy from the view of FAM134B. Nuclear 372 transcription factor Nrf2 is closely related to the level of oxidative stress. Nrf2 has 373 been proven to effectively inhibit oxidative stress in cells. So, we asked whether Nrf2 374 may regulate ER-Phagy. 375 Firstly, we use LV-Nrf2 and LV-sh-Nrf2 to overexpress or knock down the mRNA 376 expression of Nrf2. According to the RT-PCR results, we found that when LV-Nrf2 is 377 used to overexpress the level of Nrf2 mRNA level in NPCs, the mRNA level of 378 FAM134B will also be further upregulated. When Nrf2 was knocked down by 379 LV-shNrf2, FAM134B mRNA will reduce (Fig. 5A). Then, we use western blot to 380 verify the effect of LV-shNrf2 and LV-Nrf2 shows that LV-shNrf2 can knock down the 381 protein levels of total and nuclear Nrf2, and after using LV-Nrf2, the protein levels of 382 total and nuclear Nrf2 are significantly increased. It is proved that lentivirus can 383 effectively regulate the protein expression of Nrf2 (Fig. 5B&C) when Nrf2 over-expression, FAM134B can be up-regulated (Fig. 5B&C). Therefore, 387 we have reason to believe that Nrf2 has the potential to regulate FAM134B. Finally, 388 we used a confocal microscope to observe the changes in the phagocytic level of 389 NPCs after the lentivirus overexpressed Nrf2 or knocked down Nrf2 under 390 physiological condition or TBHP treatment (Fig. 5D&E). 391 Our results show that Nrf2 can effectively regulate the expression of FAM134B, 392 thereby affecting the activity of ER-phagy. while overexpression of Nrf2 inhibited this phenomenon (Fig.6E).

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After that, we verified by western blot that the therapeutic effect induced by Nrf2 408 can be reversed after LV-shFAM134B knockdown. As expected, after knocking down 409 of FAM134B, the ER-stress and apoptosis biomarkers increased significantly, 410 comparing with LV-Nrf2 treatment group (Fig.6F-I). Immediately afterwards, the 411 immunofluorescence double staining results of CHOP (488nm)-BCL-2 (594nm) also 412 confirmed the same phenomenon (Fig. 6J). TUNEL results also confirmed the same 413 point of view (Fig.6K).

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In summary, these results indicate that Nrf2 may suppress ER-stress and apoptosis 415 through FAM134B mediated ER-phagy. in IDD in vivo, we injected lentivirus into NP tissue to regulate the expression of 422 FAM134B and Nrf2, and assess its effects on IDD progress in rats.

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The intervertebral discs were evaluated at 0, 4, and 8 weeks after IDD surgery. We 424 used the disc height index (DHI) to evaluate the degree of IDD based on images 425 obtained by X-ray, respectively (Fig.7A, B). The height of the intervertebral disc 426 displayed by X-ray was normal, and there was no significant difference among groups  (488nm)-BCL-2 (594nm) also confirmed the same phenomenon (Fig.7F). 447 These results demonstrate that overexpression of FAM134B and Nrf2 could 448 ameliorate IDD in rats in vivo.

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In the current study, we reported for the first time that the ER-phagy level is 452 reduced during oxidative stress. Through manipulating ER-phagy by FAM134B, we 453 found that ER-phagy may suppress ER-stress and apoptosis in NPCs. At the meantime, 454 it was also found that Nrf2 may regulate the expression of FAM134B, thus promote 455 ER-phagy in NPCs.

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Through co-localization of LAMP1 and CANX, as well as RAMP-GFP-mCherry 457 system, we found the level of ER-phagy is significantly reduced under oxidative 458 stress (TBHP stimulation) (Fig. 1) previous studies, we observed oxidative stress may also regulate ER-phagy, though it 462 may not promote, but suppress ER-phagy in NPCs. 463 Next, we explored the mechanism of the reduced ER-phagy in NPCs during

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We found that both mRNA and protein level of FAM134B was significantly 478 down-regulated during oxidative stress, and this phenomenon was also confirmed in 479 degenerative nucleus pulposus tissues in rats (Fig. 2). These results suggested that the

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The regulation of oxidative stress on ER-phagy is the main finding of our study.

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Declaration of competing interest 540 The authors declare that they have no known competing financial interests or 541 personal relationships that could have appeared to influence the work reported in this 542 paper.