Manual therapy regulates oxidative stress in aging rat lumbar intervertebral discs through the SIRT1/FOXO1 pathway

Effects of MT on the behavior of aging rats

Due to rats’ inability to accurately convey their feelings, a single behavioral assay might not precisely capture the presence of LBP in these animals. It is therefore, reasonable to use a combination of stimulation and non-stimulation to evaluate pain. The rats’ paw withdrawal threshold (PWT) can be determined through stimulation to assess their mechanical hyperalgesia. As shown in Figure 1A, the PWT of the young rats increased slowly as they aged from 0 to 6 months, an increase that might be related to the enhancement of muscle strength and adaptation to stimulation. This upward trend remained even in the aging rats; the PWT of the 12-month-old rats continued to increase until 15 months of age. From the age of 16 months, the PWT of the rats in the aging group (AG) was significantly lower than that for the previous month (P < 0.01), until it decreased to 65.00 ± 4.78 at the age of 18 months. The PWT of the manual therapy group (MTG) was significantly higher than that of the AG at the same time point after the first month of intervention (P < 0.05, P < 0.01), and the statistically significant difference remained until the age of 18 months, when the PWT of the MTG remained at 76.17 ± 5.07.

Figure 1. Effects of MT on the behaviors of aging rats. (A) The PWT of rats were obtained by stimulating to assess their mechanical hyperalgesia. Compared with the previous time point of this group, ^#P < 0.05, ^##P < 0.01; compared with the AG at the same time point, ^*P < 0.05, ^**P < 0.01. Rats in the YCG were 6 months old at BL; rats in the AG and MTG were 12 months old at BL. (B) The open field test was used to measure mobility and voluntary standing to indirectly reflect whether the rats had LBP. (C–F) Further quantitative analysis was used for the distance traveled, mean and max speed, and the standing time/ resting time of the rats. Values are expressed as the mean ±SD (N = 8). Compared with the YCG, ^##P < 0.01; compared with the AG, ^*P < 0.05, ^**P < 0.01. PWT: paw withdrawal threshold, BL: baseline, YCG: young control group, AG: aging group, MTG: manual therapy group.

The open field test was used to measure mobility and voluntary standing, which is a non-stimulating method to indirectly reflect whether rats have LBP. As shown in Figure 1B, the 6-month-old rats preferred to explore the surrounding environment and run continuously around the field; while the 18-month-old rats seemed to prefer moving slowly at the edge of the field, especially in the corners. Further quantitative analysis (Figure 1C–1F) showed that during the 5-minute test, the distance traveled, the mean and max speed, and the standing time/resting time of the AG were significantly lower than those of the young group (P < 0.01). After 6 months of continuous MT intervention, the distance traveled, the mean speed, and the standing time/resting time of the MTG were significantly higher than those of non-intervened rats of the same age (P < 0.05, P < 0.01); however, the MT intervention did not seem to improve the maximum speed of the aging rats (P > 0.05). These results revealed that the aging rats spent less time standing and exploring, which might be partially improved by the MT intervention.

MT improved degenerative changes in the lumbar IVDs with aging

To intuitively evaluate the age-related changes in the lumbar IVD, we took radiographs of the rats’ lumbar spine in the coronal and sagittal planes (Figure 2A). The X-ray images indicated that the IVDs of the AG were characterized by height loss and blurring of the CEP boundary, which can be quantified by calculating the disc height index (DHI) (Figure 2B). Compared with the 6-month-old rat lumbar IVDs, the 18-month-old lumbar IVDs showed a significant reduction in DHI (P < 0.01), indicating that MT can significantly increase the DHI of aging rats (P < 0.01) (Figure 2C).

Figure 2. MT improved degenerative changes in the lumbar IVDs with aging. (A) Radiographs showed the sagittal and coronal view of representative L5/L6 vertebrae. (B) DHI was measured on sagittal radiographs with the equation below. (C) Quantification analysis was used for DHI. (D) Histological scores of disc degeneration were analyzed. (E) The tissue sections were stained with HE and Safranin O/Fast Green (bar = 500 and 100 μm). The white arrow indicates severe degenerative phenotypes, the black arrow indicates inner layers buckling inward, and the green arrow shows partial necrosis in the disc. Values are expressed as the mean ± SD (N = 8). Compared with the YCG, ^##P < 0.01; compared with the AG, ^*P < 0.05, ^**P < 0.01. DHI: disc height index, YCG: young control group, AG: aging group, MTG: manual therapy group.

To observe the degenerative disc phenotypes, the tissue sections were stained with hematoxylin and eosin and Safranin O/Fast Green. The results showed that the young control group (YCG) had healthy discs (Figure 2E), characterized by normal cellularity with a mix of large, vacuolated (notochordal) cells in the NP, and clear demarcation between NP and AF tissues with an intact and tightly arranged structure. On the other hand, the discs of the AG revealed severe degenerative phenotypes, including cell arrangement disorders, pronounced cell loss, and the presence of non-vacuolated cells (Figure 2E, white arrow). The clear distinction between the NP and AF tissue compartments was lost, and some of the inner AF layers showed inward buckling (black arrow). Tissue demarcation between the NP and CEP was indistinct and showed partial necrosis (green arrow). Similarly, the junction between the AF and NP was blurred in the discs of the MTG due to aging. Compared with the AG, however, the rats that underwent MT had improved degenerative disc phenotypes, including cell loss, altered cell distribution and morphology, thus maintaining IVD function. The histological scores revealed (Figure 2D) that the IVD scores of the AG were significantly higher than those of the YCG (P < 0.01), while the scores for the MTG were significantly lower than those of the AG (P < 0.05).

Taken together, these data suggest that aging rats develop degenerative IVDs, resulting in structural destruction and cellular disorder, and that MT can alleviate the age-related changes in IVDs.

MT altered the redox balance of lumbar IVDs by promoting the antioxidant capacity

To assess the effects of MT on the oxidative balance of IVDs, we measured ROS levels, superoxide dismutase (SOD) activity, and the malondialdehyde (MDA) and glutathione (GSH) content. The ROS expression levels in the NP tissues of the aging rats were significantly higher than those of the young rats (P < 0.01), suggesting that aging can cause more oxidative stress damage to tissues (Figure 3). As the product of lipid peroxidation, the MDA content of the aging rats was also significantly higher than that of the young rats (P < 0.01). In contrast, SOD and GSH are important antioxidant enzymes for countering the overproduction of active free radicals. However, the SOD activity of the NP in the AG was significantly lower than (almost half) that in the YCG (58.06 ± 17.86 vs. 115.71 ± 18.20; P < 0.01). Although the GSH content did not decrease substantially (11.38 ± 0.86 vs. 13.99 ± 1.07), the difference was statistically significant (P < 0.01). The results indicated that aging decreased IVD antioxidant capacity, disrupting the original redox balance.

Figure 3. MT modified the redox balance of lumbar IVDs by promoting antioxidant capacity. (A–D) The level of ROS, the activity of superoxide dismutase SOD, and the content of MDA and GSH were detected to assess the effects of MT on oxidative balance. Values are expressed as the mean ± SD (N = 8). Compared with the YCG, ^##P < 0.01; compared with the AG, ^**P < 0.01. ROS: reactive oxide species, SOD: superoxide dismutase, MDA: malondialdehyde, GSH: glutathione, YCG: young control group, AG: aging group, MTG: manual therapy group.

The lack of antioxidant capacity and redox imbalance in the lumbar IVDs might be modified by the MT intervention. Compared with the non-intervened aging rats, the rats that underwent MT had enhanced SOD activity and GSH content and decreased ROS and MDA content, differences that were statistically significant (P < 0.01).

MT delayed NPC senescence in lumbar IVDs

To objectively evaluate the effects of MT on aging rat lumbar IVD tissue, we measured the cellular senescence-related indexes. Senescence-associated β-galactosidase (SA-β-Gal) staining showed that the blue positive cells (Figure 4B, black arrow) were densely arranged in the IVD tissues of the aging rats, compared with few positive cells in the young rats. Further statistical analysis (Figure 4B) revealed that the SA-β-Gal-positive cell rate in the IVD tissues of the young rats was significantly lower than that of the aging rats (P < 0.01). Moreover, NPC telomerase activity was indirectly reflected by the relative mRNA expression of telomerase reverse transcriptase detected by polymerase chain reaction (PCR). The results (Figure 4C) showed that the NPC telomerase activity in the AG was significantly lower than that in the YCG (P < 0.01). These two indicators can be used as relatively specific cell sensitivity markers. Using these markers, we found that MT can significantly reduce the SA-β-Gal-positive cell rate and improve telomerase activity (P < 0.01), which suggests that MT can play an important role in delaying cell aging.

Figure 4. MT retarded the senescence of NPCs in lumbar IVDs. (A) SA-β-Gal staining showed the cellular senescence in discs (bar = 100μm). The blue cells indicated by the black arrow are senescence-related positive cells. (B) The percentage of SA-β-Gal positive cells was calculated. (C) Telomerase activity of the NPCs was indirectly reflected by the relative mRNA expression of telomerase reverse transcriptase detected by PCR. (D–G) Western blot analysis showed the relative expressions of senescence-related p53, p21 and p16 proteins in NPCs. Values are expressed as the mean ± SD (N = 8). Compared with the YCG, ^##P < 0.01; compared with the AG, ^**P < 0.01. NPC: nucleus pulposus cell, SA-β-Gal: senescence-associated β-galactosidase, YCG: young control group, AG: aging group, MTG: manual therapy group.

The western blot analysis (Figure 4D–4G) showed that the expression of senescence-related p53, p21, and p16 proteins in the NPCs of the AG were significantly higher than those of the YCG (P < 0.01) and that MT can significantly lower the expression of these proteins when compared with the non-intervened aging rats (P<0.01).

Taken together, these data suggest that aging induces a decrease in NPC telomerase activity in the rat lumbar IVDs, which can activate p53, p21, and p16 proteins regulating cell senescence, thereby accelerating the aging process. MT intervention can inhibit the reduction of telomerase activity, reducing the expression of senescence-related proteins, thereby delaying NPC senescence in lumbar IVDs.

MT exerted its effects by regulating the SIRT1/FOXO1 pathway

To investigate the possible mechanism by which MT reduces lumbar IVD degeneration, we analyzed the changes in the SIRT1/FOXO1 pathway in the NPCs. Immunofluorescence staining showed that SIRT1 (Figure 5A, red fluorescent) and ac-FOXO1 (Figure 5A, green fluorescent) were co-expressed in the NPCs of the MTG (Figure 5A, white arrow). However, ac-FOXO1 was difficult to observe in the NPCs of the YCG, and there was a large amount of SIRT1. In contrast, there was only a small amount of SIRT1 in the AG, and ac-FOXO1 was mainly expressed in the NPCs. These results suggest that SIRT1, which is abundantly expressed in the NPCs of the young rats, can inhibit FOXO1 acetylation to maintain NPC homeostasis; the lack of SIRT1 in the aging rats can increase the degree of FOXO1 acetylation and disrupt the original homeostasis. The MT intervention can significantly increase SIRT1 expression in the NPCs and reduce ac-FOXO1 expression (P < 0.01), thereby reducing the effects of NPC aging (Figure 5B, 5C).

Figure 5. MT exerted its effects through regulating the SIRT1/FOXO1 pathway. (A) Immunofluorescence staining showed the expressions of SIRT1 (red fluorescent) and ac-FOXO1 (green fluorescent) (bar = 50 μm). The white arrow indicates that the two proteins were co-expressed in the NPCs. (B,C) The fluorescence intensity of SIRT1 and ac-FOXO1 was analyzed. (D–G) A Western blot analysis showed the relative expressions of SIRT1, FOXO1 and ac-FOXO1 proteins in NPCs. (H,I) PCR results showed the relative mRNA expressions of SIRT1 and ac-FOXO1. Values are expressed as the mean ± SD (N = 8). Compared with the YCG, ^##P < 0.01; compared with the AG, ^**P < 0.01. SIRT1: silent mating type information regulation 2 homolog-1, FOXO1: forkhead box O1, YCG: young control group, AG: aging group, MTG: manual therapy group.

The western blot analysis (Figure 5E) also showed changes in the SIRT1, FOXO, and ac-FOXO1 proteins in the pathway. The results for SIRT1 and ac-FOXO1 were consistent with those of the immunofluorescence (Figure 5D, 5G). Compared with the YCG, the AG had significantly decreased FOXO1 expression in the NPCs (P < 0.01), which might have been caused by acetylation of a large amount of FOXO1, and MT might indirectly increase FOXO1 expression by inhibiting acetylation (P < 0.01). In addition, the PCR results for SIRT1 and FOXO1 further confirmed the above results (Figure 5H, 5I). Thus, MT might exert its effects by regulating the SIRT1/FOXO1 pathway.

Animals

Eight young male Sprague–Dawley rats (2 weeks) and 16 adult males (12 months, weight 700 ± 50 g) were obtained from Shanghai Jihui Laboratory Animal Co., Ltd. [certificate number: SCXK (Shanghai)2017-0012] and housed in the Laboratory Animal Center of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. All the rats were fed in standard laboratory conditions (22~27° C, 50%–70% indoor humidity) under a 12-h light-dark cycle with rat chow and water ad libitum for one week before the experiment. To alleviate pain and avoid injury, the animal feeding, caring, and intervention were conducted in accordance with procedures approved by the Experimental Animal Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (ethics number: YYLAC-2020-095).

Grouping and intervention

The 16 adult male Sprague-Dawley rats were randomly divided into the aged group (AG) and the manual therapy group (MTG) (N = 8). The 8 immature male rats comprised the young control group (YCG). The MTG underwent manual therapy on the acupoint beside the spine from 12 months of age. The specific operation was as follows: 1) before the intervention, the rats were placed in the experimenters’ hands 30 min prior to the operation to adapt; (2) the right thumb was used to press and knead the Shenshu (BL23) points on the back of the rat, which are located on both sides of the second lumbar vertebrae and on the erector spinalis muscle (Figure 6); (3) to maintain the amount of stimulation during the manipulation, the right thumb was operated with a wireless tactile measurement finger sleeve (FingerTPS, Pressure Profile System, USA), with a pressure of 5 N and a frequency of 2 Hz [27]; 4) the rats underwent the intervention alternately on the left or right side of the spinal column, one acupoint each time for 10 min, once a day, 6 days a week for 6 months; and 5) after the MT intervention, the rats were pacified before being put into the cage. The AG and YCG underwent no intervention but were grabbed and fixed in the same way as the MTG.

Figure 6. Location of Shenshu (BL23) point and IVD levels used for each outcome. IVD: intervertebral disc.

Behavioral testing

Behavioral tests were conducted for 6 months during the light cycle between 9 am and 5 pm. The rats were habituated to the experimental environment for 2 h daily 3 days before testing. The mechanical hyperalgesia test was performed on the rats every month, once a day for 3 consecutive days, and the mean results of the 3 tests were taken as the final behavioral test results of the month. The open field test was performed after the 6-month intervention, repeated tests and data processing method was the same as above. The tests were implemented, recorded and supervised by 3 experimenters, respectively, and rotated after each test to reduce the experimental error as much as possible.

An electric von Frey aesthesiometer (IITC/Life Science, USA) was applied to test the mechanical hyperalgesia of the rats, as previously reported [60]. The tip head was aimed at the central part of the left hind foot of the rat and was raised at a constant speed for stimulation. When the aesthesiometer recorded the maximum amount of stimulation making the rat withdraw, and the value of this force was assigned to the paw withdrawal threshold (PWT). To prevent sensory sensitization caused by frequent stimulation, the right foot should be stimulated at least 20 s after the measurement of the left foot. The left and right feet of each rat were repeatedly measured 3 times. The maximum and minimum of the 6 values were deleted, and the mean of the remaining 4 values was deemed as the rat’s PWT in one test [61].

The open field test consisted of a black square field (100×100 cm) surrounded by 30-cm high walls made of translucent plastic, which was placed in a quiet and well-lit room. The test was initiated by placing a single rat in the center of the field and allowing it move freely for 5 min while being photographed by a camera. The field was cleaned with low alcohol after every test, and the next test was carried out after the alcohol evaporated completely. The behaviors of the rats were measured by the experimenter and by analysis software (SMART v3.0); the main parameters were as follows: (1) distance traveled: the total travel distance of the rats in the field; (2) mean speed and max speed: the average speed of the rats in the field and the maximum speed at a certain time point; (3) resting time: the time during which the rats did not move horizontally (including standing time); (4) standing time: the time of rats standing on their hind legs (including wall climbing).

Radiography and relative height of disc measurement

After the final behavioral testing, images of the lumbar spine were captured with X-ray imaging and irradiation systems (Samsung XGEO, South Korea). Digital images were obtained at 55 kVp under identical imaging conditions using identical acquisition parameters. The rats were anesthetized with 2% pentobarbital sodium and placed in the X-ray chamber on their ventral side with all limbs extended away from the midline in a natural position to obtain coronal images. The rats were then placed on their side in a natural position with the tail extended to obtain sagittal images. Disc height indices (DHI) were measured using ImagePro plus 6.0, by averaging measurements from the anterior, middle, and posterior portions of the IVD (L5/L6) from a sagittal plane and dividing it by the average measurements of the anterior, middle, and posterior portions of the adjacent vertebral bones [62].

Histological staining

After 6 months of intervention, all rats were euthanized. Intact lumbar spinal segments and IVD tissues from L5/L6 were collected and fixed in 4% formaldehyde for 48h, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 1 month. Tissues were then dehydrated in gradient alcohol and embedded in paraffin. 4 μm sections were cut and stained with either hematoxylin and eosin (HE) or Safranin O/Fast Green according to standard procedures. Images were acquired using a microscope (Leica, Germany) and were scored by 2 independent observers using the modified Thompson grading scale [63] to evaluate the degeneration of IVDs.

Detection of redox balance in IVDs

Fresh NP tissues dissected from L2-L5 were ground and homogenized then lysed thoroughly in RIPA Lysis Buffer (Beyotime, China) at a concentration of 10ul/mg. The cell lysate was centrifuged at 12,000×g for 5 min, and the concentration of the supernatant was calculated by using a BCA protein concentration assay kit (Beyotime, China). Based on the concentration, the tissue homogenate was diluted to 1 mg/ml with PBS.

Test kits (Nanjing Jiancheng, China) were used to detect the level of ROS, the activity of SOD, and the content of MDA and GSH according to the instructions. Additionally, 2, 7- dichlorofluorescein diacetate (DCFH-DA) was used as a probe for ROS, and the fluorescence intensity of ROS was measured at 488nm/525nm with a microplate reader (Thermo Scientific, USA) to determine the level of ROS.

Senescence-associated β-galactosidase activity assay

Fresh IVDs were dissected from L6/S1, directly embedded in optimal cutting temperature compound, and stored at −80° C to detect the activity of SA-β-Gal in IVDs. Next, 5 μm sections were cut and stained with SA-β-Gal kit (Beyotime, China) according to the manufacturer’s protocol. Before mounting them with resin, sections were stained again with eosin for 10 sec. The number of cells positive (blue) for localization of SA-β-Gal and total cells (blue and red) in the field were counted using the ImagePro plus 6.0 under 400× magnification. The ratio was recorded as the percentage of positive cells. Three fields were randomly selected from each section to calculate the average percentage of SA-β-Gal positive senescent cells.

Immunofluorescence

After deparaffinization and rehydration, the prepared paraffin sections were treated with citric acid buffer (0.01 mol/L, pH6.0) at 98° C for 15 min for antigen retrieval and were cooled to room temperature for 1 h, followed by incubation with 1% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. The sections were then incubated in 5% bovine serum albumin (BSA) (Beyotime, China) at room temperature for 30 min, followed by primary antibody incubation (SIRT1, 1:50, CST, 8469s; ac-FOXO1, 1:50, Affinity Biosciences, AF2305) overnight at 4° C. After 12 h, the sections were rewarmed in an oven at 37° C for 45 min and incubated with fluorescein-conjugated secondary antibodies (1:100, Sanggon Biotech, China) at room temperature for 30 min in the dark. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) for 2 min. Three fields were randomly selected from each section to calculate the average fluorescence intensity of the target protein under 400× magnification.

Western blot

The extraction protocol for total protein quantification of fresh NP tissues was described above. After adjusting the protein concentration, 20 μg tissue lysates were separated by 12% SDS-PAGE gel using electrophoresis and transferred to PVDF membranes. The membranes were incubated with primary antibodies (p53, 1:1000, Abcam, ab26; p21, 1:1000, Abcam, ab109520; p16, 1:1000, Abcam, ab51243; p53, 1:1000, Abcam, ab26; SIRT1, 1:1000, CST, 8469s; FOXO1, 1:1000, CST, 2880s; ac-FOXO1, 1:1000, Affinity Biosciences, AF2305; GAPDH, 1:5000, CST, 5174s) overnight at 4 after blocked by 5% BSA at room temperature for 2 h. The membranes were rinsed 3 times with TBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, Sanggon Biotech, China) at room temperature for 2 h. The final results were acquired using the ChemiDoc MP system (Bio-Rad, USA) with enhanced chemiluminescence (ECL) kit (Millipore, USA) and were quantified using the ImagePro plus 6.0.

Quantitative real-time PCR

Reverse transcription was performed with a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Bio, Japan). Real-time polymerase chain reaction (RT-PCR) was performed on a PCR System (Bio-Rad, USA) with SYBR Premix Ex Taq (TaKaRa, Japan). The reaction conditions were a single cycle of 95° C for 5 min, and 40 cycles of 95° C for 15 sec, 60° C for 30 sec. GAPDH was used as a reference, each sample was tested in triplicate. The relative expression of the target gene was calculated using the 2^-ΔΔCt method. The primers used in the study are shown in Table 1.

Statistical analysis

SPSS 21.0 software was used for the statistical analysis. Normality and homogeneity of variance tests were performed on the data. The data are presented as mean ± standard deviation (SD) if they were normally distributed; data that did not conform to the normal distribution are presented as the median (P25 and P75). If the data conformed to the normal distribution, the comparison of differences among groups was performed using one-way ANOVA. If the variances were homogeneous, a pairwise comparison was performed using the least significant difference test; if the variances were not homogenous, Dunnett’s T3 test was used for pairwise comparison. If the data did not conform to the normal distribution, a nonparametric Kruskal-Wallis H test were performed and using the Nemenyi method for pairwise comparison. Repeated measurement analysis of variance was used to analyze the data on the same observation index at various time points between groups. If the results were statistically different, a multivariate analysis of variance was used for pairwise comparison. The significance level of the statistical examination was α = 0.05. Therefore, P <0.05 indicated a significant difference.