SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy

Results

SIRT1 expression decreased in NP tissues with intensified IVDD

First, we demonstrated the relationship between SIRT1 expression and cellular senescence in human NP tissues. The degenerative grades of the lumbar discs from patients undergoing spine surgery were evaluated according to the Pfirrmann classification [35], and representative preoperative magnetic resonance imaging (MRI) scans of donor IVDs with different degrees of degeneration are shown in Figure 1A. SA-β-Gal staining was used to detect the degree of senescence of NP cells derived from NP tissues of patients with different grades of degenerative discs. The results showed that the proportion of SA-β-Gal-positive cells and staining intensity significantly increased with intensified IVDD (Figure 1B). Immunohistochemistry staining of patient NP tissues showed that SIRT1 was expressed in all tissues, whereas the expression level gradually decreased as the disc degeneration intensified (Figure 1C). These results indicated that the expression of SIRT1 was reduced, but the extent of cellular senescence was enhanced as intervertebral disc degeneration progressed.

Figure 1. Detection of SIRT1 expression and cellular senescence in human degenerative NP tissues. (A) Preoperative lumbar MRI images of patients’ IVD tissues were classified via Pfirrmann’s grading system. Degenerative IVDs scored III-IV were defined as early stage of IVDD, and the discs scored V were defined as advanced stage of IVDD. Red arrows refer to the position of the acquired NP tissue. (B) The degree of senescence of NP cells isolated from IVDs with different grades of degeneration was detected and analyzed using SA-β-Gal staining (200×). (C) The expression level of SIRT1 in NP tissues obtained from patients with different Pfirrmann grades was detected by immunohistochemistry (200×). *P<0.05 versus the Pfirrmann III group. ^#P<0.05 versus the Pfirrmann IV group. Black bars=100 μm.

High-magnitude compression exacerbated cellular senescence and weakened SIRT1 expression in NP cells

For further analysis of the relationship between SIRT1 expression and cellular senescence in the process of compression-induced NP degeneration, human NP cells seeded in the Gel-MA scaffold were perfusion-cultured with dynamic high-magnitude compression (20% compressive deformation) using our self-developed perfusion bioreactor (Figure 2A). As is shown in Figure 2B, the percentage of SA-β-Gal-positive cells (green), and the staining intensity significantly increased in the NP cells subjected to high-magnitude compression. The expression of senescence-related biomarkers tested by Western blotting revealed that AMPK decreased, whereas P53, P21 and P16 were strongly upregulated in NP cells that experienced high-magnitude compression, which was consistent with results of the SA-β-Gal staining (Figure 2D). The immunohistochemistry data showed that the expression level of SIRT1 substantially decreased in the NP cells under high-magnitude compression (Figure 2C). Additionally, Western blotting results indicated that SIRT1 and PGC-1α were downregulated by high-magnitude compression in human NP cells (Figure 2D); PGC-1α is a marker of the deacetylation activity of SIRT1 [36].

Figure 2. Analysis of SIRT1 expression and the degree of senescence in human NP cells subjected to high-magnitude compression. (A) Schematic of the primary units of the substance exchanger-based perfusion bioreactor system: (1) medium reservoir; (2) peristaltic pumps; (3) tissue culture chambers; (4) substance exchanger; (5) pH, PO₂ and PCO₂ sensor; (6) loading application devices. NP cell-encapsulated Gel-MA hydrogels were dynamically compressed by the loading application devices in the chambers at 0% (a) and 20% (b) compressive deformation for the control and high-compression groups, respectively at a frequency of 1.0 Hz. (B) The degree of senescence of the NP cells subjected to 0% or 20% (high-magnitude) compressive deformation was analyzed using SA-β-Gal staining (200×). (C) The expression level of SIRT1 in the NP cell-encapsulated hydrogels was detected by immunohistochemistry (200×). (D) The SIRT1, PGC1α and senescence-related protein (AMPK, P53, P21 and P16) levels were analyzed by Western blotting. *P<0.05 versus the control group. Black bars=100 μm.

High-magnitude compression triggered oxidative stress injury and impaired mitochondrial function in NP cells

Our previous study confirmed that high-magnitude compression could accelerate NP cell senescence through the ROS pathway [11]. As the major ROS-generating organelle, mitochondria are also the main target of oxidative stress injury. Thus, we utilized flow cytometry and immunofluorescence to directly and indirectly analyze the content of intracellular ROS in NP cells stained with 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe, and the data revealed that high-magnitude compression increased the ROS content in NP cells (Figure 3A). Additionally, the mitochondrial membrane potential of the NP cells of each group was detected using JC-1 fluorescent probe staining. The results of immunofluorescence and flow cytometry revealed that the green/red fluorescence ratio of NP cells treated with high-magnitude compression strongly increased, indicating a depressed state of mitochondrial membrane potential (Figure 3B). These data suggested that high-magnitude compression could cause ROS accumulation and trigger mitochondrial dysfunction in NP cells.

Figure 3. Measurement of ROS content and mitochondrial membrane potential in the NP cells with or without Ad-SIRT1 treatment under high-magnitude compression. (A) After high-compression or high-compression plus Ad-SIRT1 treatment, the ROS level in NP cells was observed and measured by fluorescence microscopy (200×) and flow cytometry following DCFH-DA fluorescent probe staining. (B) The mitochondrial membrane potential of NP cells was observed and measured by fluorescence microscopy (200×) and flow cytometry after JC-1 fluorescent probe staining. The difference in mitochondrial membrane potential is presented by the green/red fluorescence intensity ratio. *P<0.05 versus the control group. ^#P<0.05 versus the high-compression group. White bars=100 μm.

High-magnitude compression caused mitochondrial injury and depressed mitophagy in NP cells

Mitophagy is found to be a type of mitochondria-specialized autophagy that can selectively remove injured mitochondria to maintain mitochondrial homeostasis [37]. Hence, we hypothesized that there may be a connection between compression-induced mitochondrial dysfunction and mitophagy in NP cells. As is shown in Figure 4A, the NP cells subjected to the high-compression conditions revealed an increase in the amount of nuclear GFP-LC3 puncta, which is a sign of inhibited autophagy. In addition, results of Western blotting also revealed that the expression of SIRT1 and the ratio of LC-3II/I decreased in the high-compression-treated group, indicating that autophagy was retarded (Figure 4B). In addition, an immunofluorescence assay was used to measure the expression level of the mitophagic marker PINK1, and an obvious inhibition of PINK1 expression was found in NP cells subjected to high-magnitude compression (Figure 4C). Furthermore, TEM observation of the morphology of mitochondrial revealed that mitochondria in the compression-treated NP cells appeared smaller than normal with increased membrane density compared with those in the normal NP cells (Figure 4D). In addition, TEM observation of autophagosomes, smooth vacuoles surrounded by a double-membrane without ribosomes, revealed that high-magnitude compression-treated NP cells showed a decreased number of autophagosomes (Figure 4D). The data above further proved that high-magnitude compression could lead to mitochondrial injury, which may involve abnormal mitophagy.

Figure 4. Observation of mitophagy in human NP cells with or without Ad-SIRT1 treatment under high-magnitude compression. (A) The distribution of GFP-LC3 puncta in NP cells subjected to high-compression or high-compression plus Ad-SIRT1 treatment was visualized by confocal microscopy (400×) after transfection of GFP-LC3-expressing adenovirus. (B) The relative expression levels of SIRT1 and LC3II/I in NP cells subject to high-compression or high-compression plus Ad-SIRT1 treatment were analyzed by Western blotting. (C) The expression of the key mitophagic regulator PINK1 was observed and analyzed by fluorescence microscopy (400×) after immunofluorescence staining. (D) The electron micrographs of mitochondria and autophagosomes in NP cells were observed using TEM (5000×), and double-membrane profiles resembling pieces of mitochondria could be found in some autophagosomes in the Ad-SIRT1-treated group. Blue arrows show the typical autophagic vacuole double-layer ultrastructural morphology. Red arrows represent the electron micrographs of mitochondria in the NP cells from each group. *P<0.05 versus the control group. ^#P<0.05 versus the high-compression group. White bars=100 μm. Black bars=2 μm.

Upregulation of SIRT1 activated mitophagy and alleviated mitochondrial injury in human NP cells under high-magnitude compression.

Previously, we speculated that SIRT1 may protect against compression-induced senescence in NP cells by regulating mitophagy, due to its antioxidant and mitophagy-acting properties. For confirmation of this hypothesis, SIRT1 was upregulated using Ad-SIRT1, and the status of mitophagy and mitochondrial function in NP cells subjected to high-magnitude compression were analyzed. As is shown in Figure 3A, 3B, treatment with Ad-SIRT1 significantly attenuated the ROS content and alleviated the abnormal state of mitochondrial membrane potential in the NP cells subjected to high-compression. Notably, in NP cells, the switch to high-compression conditions resulted in an increase in the amount of nuclear GFP-LC3 puncta, whereas distribution of GFP-LC3 translocated from the nucleus into the cytoplasm when the NP cells were treated with Ad-SIRT1 (Figure 4A). Moreover, Western blotting analysis and immunofluorescence observation showed enhanced expression levels of SIRT1, LC3-II/I and PINK1 in the SIRT1-overexpressing NP cells compared with the other groups of cells (Figure 4B, 4C). Additionally, TEM observation showed a substantially increased number of autophagosomes in the Ad-SIRT1-treated NP cells compared with the NP cells from the control and high-compression-treated groups (Figure 4D). TEM observation also found fragmentations that resemble mitochondrial sections being engulfed by double-layer vesicles in Ad-SIRT1-treated NP cells, which is an indicator of mitophagy (Figure 4D).

Blocking of mitophagy attenuated the anti-senescence role of SIRT1 in NP cells under high-magnitude compression.

To further clarify that SIRT1 protects against compression-induced senescence of NP cells by inducing mitophagy, we transfected NP cells with PINK1-shRNA to silence the endogenous PINK1 expression before compression treatment. Next, the expression levels of senescence-related proteins (AMPK, P53, P21, P16) were analyzed using Western blotting, and the data showed that the expression levels of AMPK decreased, whereas P53, P21 and P16 increased in the high-magnitude compression+PINK1-shRNA-treated group (Figure 5A). In addition, administration of Ad-SIRT1 significantly enhanced the expression level of AMPK and attenuated the expression of P53, P21 and P16 in the NP cells under high-magnitude compression, but this treatment did not have the same effect in the PINK1-knockout NP cells (Figure 5A). Moreover, the results of SA-β-Gal staining also revealed that the NP cells treated with high-magnitude compression following administration of PINK1-shRNA showed the highest proportion of positively stained cells. In contrast, the Ad-SIRT1-treated NP cells had the lowest proportion of SA-β-Gal-positive cells as well as the weakest staining intensity (Figure 5B). Nevertheless, knockout of PINK1 inhabited the protective role of SIRT1 against high-magnitude compression-induced senescence in NP cells, which is consistent with the Western blotting results (Figure 5B). Next, we further analyzed the expression differences of mitophagic proteins in each group by using Western blotting. The results revealed that the expression of PINK1 and LC-3 II/I was strongly attenuated, but the expression of Parkin and P62 was enhanced in the NP cells treated with PINK1-shRNA transfection, which indicated that mitophagy was inhibited in the PINK1-knockout NP cells (Figure 5C). Furthermore, treatment with Ad-SIRT1 enhanced the expression of PINK1 and LC-3 II/I, and reduced the expression of Parkin and P62 in NP cells, indicating enhanced activation of mitophagy (Figure 5C). However, when PINK1 was knocked out in advance, the mitophagy-stimulating role of SIRT1 was also attenuated (Figure 5C). These results implied that SIRT1 could alleviate high-magnitude compression induced senescence of human NP cells by activating mitophagy.

Figure 5. Effect of SIRT1-overexpression on high-compression treated NP cells under inhibition of mitophagy. (A) PINK1-shRNA was used to silence the endogenous PINK1 in NP cells before high-compression and Ad-SIRT1 treatments. The expression levels of senescence-related biomarkers (AMPK, P53, P21 and P16) in each group were analyzed by Western blotting. (B) The degree of senescence of the NP cells in each group was assessed by optical microscopy after SA-β-Gal staining (200×). (C) The expression levels of mitophagic biomarkers (PINK1, Parkin, LC3II/I and P62) were analyzed by Western blotting. *P<0.05 versus the high-compression group. ^#P<0.05 versus the high-compression+Ad-SIRT1 group. Black bars=100 μm.

Discussion

IVDD is considered to be the most common pathological basis for spine degenerative disease, which is the major etiological factor of low back and leg pain [1]. The progressive degradation of ECM is the main pathological characteristic during IVDD [6]. NP cells are responsible for the synthesis of ECM within the NP region of the IVD; hence, the accumulation of senescent NP cells is regarded as a direct reason for the compromised matrix synthesis [3, 4]. However, previous researchers did not find a direct relationship between the patient’s age and senescent cell accumulation [3, 4, 38]. Compared with that in degenerative IVD, the expression of senescence-related biomarkers was not significantly upregulated in normal age-matched IVD [3, 4, 38]. Thus, these findings indirectly suggest that the senescent phenotype in degenerative IVD closely resembles stress-induced senescence caused by extrinsic stresses instead of the intrinsic aging process. Therefore, further investigation of the pathomechanism behind NP cell senescence and the development of strategies to retard the progression of NP cell senescence will be helpful for the treatment of spine degenerative diseases.

Human IVD mainly performs three primary functions: (a) resists compressive and tension forces during movement, (b) provides flexibility to the spine, and (c) maintains space between vertebral bodies, thereby preventing compression or injury to the spinal nerves [39]. Thus, it is understandable that NP tissue suffers various types of mechanical compression under in vivo conditions. Established studies have demonstrated that un-physiological and high compressive stress can aggravate NP cell death and exacerbate disc degeneration in animal model [8, 40]. Hence, we speculated that inhibition of high compressive stress-induced senescence of NP cells may play a vital role in retarding IVDD. Previously, we have demonstrated that high-magnitude compression could accelerate the premature senescence of NP cells, and the mechanism involved in this process was associated with oxidative stress and the ROS pathway [11]. As the major regulator of ROS generation and redox homeostasis, mitochondrial have been widely reported to be associated with NP degeneration [18, 19]. Thus, in the present study, we further detected the mitochondrial function of the NP cells subjected to high-magnitude compression by analyzing ROS content, measuring mitochondrial membrane potential and observing mitochondrial micromorphology under TEM. The experimental results indicated that high-magnitude compression caused ROS accumulation and induced mitochondrial dysfunction in NP cells.

SIRT1 has been widely reported to play a protective role against NP degeneration via multiple pathways [25, 26, 41]. The present research also revealed that the expression of SIRT1 in clinical NP samples gradually decreased, whereas the extent of cellular senescence was enhanced as disc degeneration intensified. Moreover, upregulation of SIRT1 substantially alleviated the content of intracellular ROS content and senescence of NP cells that were perfusion-cultured and underwent high-magnitude compression in vitro. Recent studies have noted that SIRT1 performs its anti-senescence and antioxidant functions by regulating mitophagy, which is a mitochondrion-protective process [27–30]. Our experimental data indicated that high-magnitude compression depressed the mitochondrial membrane potential in NP cells, whereas upregulation of SIRT1 considerably elevated the mitochondrial membrane potential to the normal state in NP cells subjected to high-magnitude compression. In fact, the variety of mitochondrial membrane potentials indirectly reflects the activation of mitophagy [42]. Mitophagy is a type of mitochondria-targeted autophagy that ameliorates various types of stress-induced cellular damage by recycling injured mitochondria [37, 43]. Additionally, established studies have verified the beneficial role of mitophagy in NP cell survival [33, 34]. Notably, another study also elucidated the relationship between SIRT1-induced autophagy and nutrient deprivation-induced mitochondrial apoptosis of NP cells [44]. Additionally, as an NAD-dependent deacetylase, SIRT1 could affect mitophagy via deacetylation of the key autophagic protein LC3 in the nucleus [28, 45]. In view of the published studies and our experimental data, we further speculated that SIRT1 exerted beneficial effects through attenuation of compression-induced senescence by regulating mitophagy. To verify this hypothesis, we used PINK1-shRNA to silence the endogenous PINK1 expression before compression and SIRT1-overexpressing treatment, because the PINK1-Parkin axis is regarded as the core machinery for mitophagy [46]. Then, the degree of senescence and mitophagic activity in NP cells were analyzed. Analysis of the senescent cells and senescence-related biomarkers suggested that deletion of PINK1 substantially exacerbated senescence in NP cells under compression stress, whereas treatment with Ad-SIRT1 effectively attenuated the compression stress-induced senescence of NP cells. However, SIRT1-overexpressing did not reverse the degree of senescence in the PINK1-knockout NP cells suffering high-magnitude compression, indicating that the lack of PINK1 blocked the protective property of SIRT1 against compression stress-induced senescence in NP cells. Furthermore, analysis of mitophagy-related proteins revealed that treatment with Ad-SIRT1 significantly enhanced the expression of PINK1 and an autophagic marker (LC3II/I), but decreased the expression levels of Parkin and P62. This finding can be explained as follows: when the process of mitophagy is activated, full-length PINK1 (66 kDa) is modified into a cleaved product (55 kDa), which translocates on the outer surface of the targeted mitochondrion and recruits the Parkin from the cytosol [43, 46]. Recruited Parkin protein combines with LC3II and then initiates the process of mitophagy to clear the targeted mitochondrion [46]. Nevertheless, in the normal mitochondrion, full-length PINK1 is released into the cytosol and then degraded by mitochondrial proteases and proteasomes [43, 46]. TEM observation also found a markedly increased number of autophagosomes and signs of mitophagy (autophagosome-engulfed mitochondrial sections) in the Ad-SIRT1-treated NP cells. Therefore, our study concludes that SIRT1 can alleviate mechanical stress-induced senescence of human NP cells under high-magnitude compression by activating mitophagy.

Although there are some novel findings in the present study, several limitations also exist. First, the NP cells used in the study were cultured under normoxic conditions, because of the lack of hypoxia-culture settings in our bioreactor. This condition is different from the physiological hypoxic condition in which the NP cells live. If possible, we will update the equipment to obtain similar hypoxic microenvironments in future studies. Second, autophagy is a quite complex and controversial process in regulating cell survival, and the present study also found that SIRT1 deacetylase could regulate the autophagy level, and knockout of PINK1 did not completely inhibit the activation of autophagy in NP cells. Thus, further research on the SIRT1-induced autophagy involved in the pathomechanism of NP degeneration is needed in the future. To conclude, our study reveals that SIRT1 expression is associated with the progression of IVDD and is also involved in the regulation of mechanical stress-induced damage to the mitochondrial and senescence in human NP cells. Moreover, upregulation of SIRT1 can effectively suppress the generation of ROS and protect against senescence in NP cells subjected to high-magnitude compression by recycling injured mitochondria by mitophagy (Figure 6). This study helps us better understand the regulatory role of SIRT1 in the process of NP cell senescence and provides more substantial evidence for the usage of SIRT1-targeting agents in the treatment of IVDD.

Figure 6. Schematic diagram shows the potential mechanism of SIRT1 overexpression. High-magnitude compression triggered mitochondrial dysfunction and induced NP cell senescence. Overexpression of SIRT1 could alleviate compression-induced senescence by activating mitophagy to eliminate injured mitochondria and retard ROS accumulation.

Author Contributions

Conception and design: Pei Li and Qiang Zhou; (II) Experiments performing: Yiyang Wang, Haoming Wang, Yunyun Zhuo, Yanzhu Hu, Zetong Zhang; (III) Manuscript writing: Yiyang Wang; (IV) Data analysis: Jixing Ye, Liehua Liu, Lei Luo, Chen Zhao; (V) Final approval of manuscript: all authors.

Acknowledgments

We thank Dr. Yibo Gan and Dr. Weikang Zhao for their generous technical assistance, and the laboratory services provided by the Clinical Research Center of the Third Affiliated Hospital, Chongqing Medical University.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This work was supported by grants from the National Key R & D Program of China (No.2018YFC1105803); Natural Science Foundation of China (No.81772378, No.81974346); Technological Innovation and Application Program of Chongqing, China (No.cstc2019jscx-msxm0606).

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