# Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway

Authors: Bo Li; Ze-Yu Lu; Sheng-Dan Jiang; Lei-Sheng Jiang; Xin-Feng Zheng
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Background. Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods. Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results. Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion. Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.


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## Body

## 1. Introduction

Intervertebral disc degeneration disease (IVDD) is one of the most common causes of disability in aging people and a decline in the quality of their life [1–4]. However, the pathogenesis of IVDD is not fully understood. A decrease in the number of live cells in the disc and reduced intracellular matrix with altered components are the pathological basis of disc degeneration [2, 5]. Increased apoptosis of disc cells is the primary reason for the decrease in the number of live cells [2, 5, 6]. Excessive apoptosis could also lead to a decrease in cell density and an increase in intracellular matrix catabolism, resulting in disc degeneration [7–9]. Thus, suppressing the excessive apoptosis of disc cells is believed to be a potential way to delay disc degeneration.Smad7 is a key member closely related to TGF-β functions. It binds to the TGF-β receptor, inhibits the phosphorylation of Receptor-Smad complex, and blocks the signal transduction of TGF-β. Meanwhile, TGF-β could induce Smad7 expression through the activation of Receptor-Smads, thus forming a positive feedback regulation. Activated Receptor-Smads could also upregulate Smad7 expression through the activation of Smad7 promoter, forming another regulatory feedback loop [10]. By blocking TGF-β signaling pathways, Smad7 inhibited the synthesis of collagen and proteoglycans. It has been reported that adenovirus with Smad7 cDNA transfected into hepatic stellate cells or renal interstitial fibroblasts inhibited the synthesis of collagen and proteoglycan and stopped the process of fibrosis [11, 12]. Our previous study also has demonstrated that Smad7 plays key roles in disc degeneration. Smad7 was found to be highly expressed in degenerated intervertebral discs in rats, and Smad7 expression was closely associated with the metabolism of the intracellular matrix [13]. It has been reported that Smad7 promoted apoptosis by blocking the NF-κB/p65 pathway in glomerular podocytes [14]. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear.IL-1β is significantly increased in degenerative intervertebral discs (IVDs) [15]. An increasing number of studies have proved that IL-1β is involved in the inflammatory response in the process of intervertebral disc degeneration. Exposure of human AF cells to IL-1β can significantly increase apoptosis [16, 17]. Our previous study also confirmed that IL-1β is involved in IVDD and IL-1β enhances the effect of serum deprivation on rat AF cell apoptosis [18]. IL-1β was reported to induce the expression of Smad7 in chondrocyte [19]. But it is still unclear whether Smad7 participates in IL-1β-induced AF cell apoptosis.Based on the above-mentioned findings, we continue our study on the role ofSmad7 in disc degeneration. In this study, we further investigated the expression of SMAD7 in human degenerative discs and clarified the role of Smad7 in the IL-1β-induced apoptosis of rat AF cells. The underlying signaling pathways of apoptosis are also explored. We hope our results will provide a new idea for the prevention and treatment of IVDD.


## 2. Materials and Methods

### 2.1. Human Disc Sample Collection

Human disc tissues were collected from patients who underwent operations for lumbar spine diseases. A total of 45 segments of intervertebral disc tissue were collected. According to Pfirrmann’s grading criteria on MRI, 4 discs were grade I, 10 were grade II, 11 were grade III, 11 were grade IV, and 9 were grade V. These 45 discs were divided into 2 groups: nondegenerative (grades I-II) and degenerative (grades III-V). The annulus fibrosus, nucleus pulposus, and endplate tissues of each disc were carefully separated in the operation table and appropriately stored for the following experiments. Human AF tissue collection and animal experiments were approved by the Ethics Committee of our university hospital (No. XHECD2015107). All experiments involving human specimens followed the Helsinki declaration.


### 2.2. Isolation and Culture of Rat AF Cells

The whole lumbar spine segments were dissected from eight healthy 3-month-old male Sprague–Dawley rats after sacrifice by intraperitoneal injection of over-dosed Sumianxin. L1/2, L2/3, L3/4, L4/5, and L5/6 discs were separated. AF cells were isolated and cultured according to our previous method [18, 20]. First-passage cells maintained in a monolayer were used throughout the experiments [21]. The isolated primary AF cells were verified by the characteristics of their cell morphology [22].


### 2.3. Adenovirus-Mediated Gene Transfer into AF Cells

The adenovirus vector withSmad7 gene overexpression was constructed by the manufacturer (Gene Pharma, China). On the day of transfection, AF cells were seeded into 24-well culture dishes at a density of 1×105 cells/well. When they reached 50% confluence, cells were transfected with the Smad7 overexpression adenovirus or negative control adenovirus at a multiplicity of infection of 100, and cells without transfection were used as control. The efficacies of transfection were determined by western blotting and real-time PCR.


### 2.4. Small Interfering RNA Transfection

Smad7 small interfering RNAs were obtained from (Gene Pharma, China). si-Smad7-1, si-Smad7-2, and si-Smad7-3 sequences were listed as follows: si-Smad7-1: 5′-AGGCAUUCCUCGGAAGUCATT-3′; si-Smad7-2: 5′-GCAGCCTAACCAGACCTTT-3′; si-Smad7-3: 5′-GGCTGGAGGTCATCTTCAA-3′. Briefly, si-Smad7 was added to OPTI medium (Gibco, USA) and then lipofectamine 3000 (Invitrogen, Carlsbad, California, CA) was added to OPTI medium (Gibco, USA) containing si-Smad7. After being incubated for 20 min, the mixtures were added to AF cells. Si-NC was served as a negative control. The efficacies of transfection were determined by western blotting and real-time PCR.


### 2.5. Quantitative Real-Time-PCR

Total RNA was obtained from AF cells using an RNA isolation kit (Invitrogen) according to the manufacturer’s protocol. cDNA was generated by using the cDNA synthesis kit (Roche). A qRT-PCR method based on SYBR Green detection was performed for gene expression quantification using the primers. The primers were as follows: humanSMAD7 forward: 5′-CGGAAGTCAAGAGGCTGTGT3′, reverse: 5′-TGGACAGTCTGCAGTTGGTT-3′; Rat Smad7 forward: 5′-AGGCATTCCTCGGAAGTCAA-3′, reverse: 5′-TGGACAGTCTGCAGTTGGTTTG-3′. Real-time-PCR was performed with ABI StepOne Plus equipment. GAPDH was used as an internal control to normalize the expression levels of different genes. The relative complementary DNA ratio was calculated using the value of threshold cycles (Ct). The amplification efficiency between the target and the reference control GAPDH was compared by using the 2–ΔΔCt method.


### 2.6. Western-Blot Analysis

The AF cells were collected at 12, 24, and 48 h after recombinant adenovirus infection. The whole cell lysis was extracted using Cell Lysis Buffer for Western and IP (Beyotime, P0013) added with PMSF (1 : 100) [23]. The relative protein levels were calculated versus GAPDH. The primary antibodies used in this study were as follows: GAPDH, Bcl-2, Bax, Bcl-XL, Cyt-c, procaspase 9, cleaved-caspase 9, procaspase 3, cleaved-caspase 3, Smad7 (Proteintech, USA, 1 : 1000), Smad4 (Proteintech, USA, 1 : 1000), total-smad2 (Proteintech, USA, 1 : 1000), total-smad3 (Proteintech, USA, 1 : 1000), P-smad2 (CST, USA, 1 : 1000), and P-smad3 (CST, USA, 1 : 1000). Following incubation with a primary antibody, the membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit or anti-goat second antibody. The membranes were then washed thrice, and the immunoreactive bands were visualized using NBT/BCIP as a substrate. Densitometric analysis was done using the NIH ImageJ Software to quantify the protein present in the detected bands. GAPDH content was assayed as standardization of sample loading. Quantitative densitometric values of each protein of interest were normalized to GAPDH.


### 2.7. Nuclear Staining Using Hoechst 33258

Three groups of AF cells were seeded in 96-well culture plates and added with Hoechst 33258 dye solution (Beyotime, C0003) followed by removing the culture media at 12, 24, and 48 h, respectively. The procedures were done as previously described [21]. After washing with PBS twice, AF cells were added with 0.5 ml 4% formaldehyde solution and fixed at RT for 10 min. Then, AF cells were washed with PBS for 2-4 times and incubated with Hoechst 33258 solution for 20-60 min in the dark at RT. Cells were observed under a fluorescence microscope under UV light followed by washing with PBS and adding with antifade reagent.


### 2.8. Cell Apoptosis Assay Using Flow Cytometry

AF cells overexpressingSmad7 were first collected for apoptosis assay using flow cytometry at 12, 24, and 48 h, respectively. And then, we used LEHD-FMK to confirm the involvement of the mitochondria-mediated pathway in the apoptosis of rat AF cells after 48 h of Smad7 overexpression. The experiments were performed according to the manual of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen TM) [7]. In brief, AF cells were collected by centrifuge at 1000 g for 5 min followed by wash with PBS and digestion using Trypsin-EDTA. After resuspension in PBS, cell numbers were counted and 5-10×104 cells were aliquoted for staining with Annexin V-FITC solutions for 10 min in the dark at RT. Propidium iodide (PI) was then added to the AF cells. All the stained cells were analyzed using a flow cytometry platform for Annexin V-FITC and PI.


### 2.9. Suppression of Apoptosis by Z-LEHS-FMK

Z-LEHD-FMK (Caspase 9 inhibitor) was purchased from MedChemExpress (MCE), and the Cat. No. is HY-P1010.AF cells were seeded in 12-well culture plates at a density of5×105 cell/plate. All the plates were divided into four groups, with three plates each group. When the confluences of cells reached 80-90%, cell culture media were changed. The cells in one group were pretreated with 6 μl Z-LEHD-FMK for 2 h after being infected with Smad7 overexpression adenovirus. The other three groups were infected with Smad7 overexpression adenovirus, empty adenovirus, or nothing. Cells were collected after 48 h to test apoptosis rate.


### 2.10. Immunofluorescence Analysis

AF cells were seeded on sheet glasses in plates. AF cells were fixed by 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked by 5% BSA (bovine serum albumin) for 1 h. The AF cells were incubated with p-Smad3 (rabbit monoclone antibody, 1 : 200, Abcam) and p-Smad2 (rabbit monoclone antibody, 1 : 400, CST) primary antibodies overnight at 4°C. AF cells were washed with PBST thrice. Finally, AF cells were incubated with Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L). Photographs were taken by Olympus BX51.


### 2.11. Statistical Analysis

All data were analyzed using SPSS11.5 statistical software. Comparisons of data between groups were analyzed using one-way ANOVA, Kruskal-WallisH-test, or independent-samples t-test according to the distribution of data and homogeneity of variance. p values less than 0.05 were considered statistically significant.


## 2.1. Human Disc Sample Collection

Human disc tissues were collected from patients who underwent operations for lumbar spine diseases. A total of 45 segments of intervertebral disc tissue were collected. According to Pfirrmann’s grading criteria on MRI, 4 discs were grade I, 10 were grade II, 11 were grade III, 11 were grade IV, and 9 were grade V. These 45 discs were divided into 2 groups: nondegenerative (grades I-II) and degenerative (grades III-V). The annulus fibrosus, nucleus pulposus, and endplate tissues of each disc were carefully separated in the operation table and appropriately stored for the following experiments. Human AF tissue collection and animal experiments were approved by the Ethics Committee of our university hospital (No. XHECD2015107). All experiments involving human specimens followed the Helsinki declaration.


## 2.2. Isolation and Culture of Rat AF Cells

The whole lumbar spine segments were dissected from eight healthy 3-month-old male Sprague–Dawley rats after sacrifice by intraperitoneal injection of over-dosed Sumianxin. L1/2, L2/3, L3/4, L4/5, and L5/6 discs were separated. AF cells were isolated and cultured according to our previous method [18, 20]. First-passage cells maintained in a monolayer were used throughout the experiments [21]. The isolated primary AF cells were verified by the characteristics of their cell morphology [22].


## 2.3. Adenovirus-Mediated Gene Transfer into AF Cells

The adenovirus vector withSmad7 gene overexpression was constructed by the manufacturer (Gene Pharma, China). On the day of transfection, AF cells were seeded into 24-well culture dishes at a density of 1×105 cells/well. When they reached 50% confluence, cells were transfected with the Smad7 overexpression adenovirus or negative control adenovirus at a multiplicity of infection of 100, and cells without transfection were used as control. The efficacies of transfection were determined by western blotting and real-time PCR.


## 2.4. Small Interfering RNA Transfection

Smad7 small interfering RNAs were obtained from (Gene Pharma, China). si-Smad7-1, si-Smad7-2, and si-Smad7-3 sequences were listed as follows: si-Smad7-1: 5′-AGGCAUUCCUCGGAAGUCATT-3′; si-Smad7-2: 5′-GCAGCCTAACCAGACCTTT-3′; si-Smad7-3: 5′-GGCTGGAGGTCATCTTCAA-3′. Briefly, si-Smad7 was added to OPTI medium (Gibco, USA) and then lipofectamine 3000 (Invitrogen, Carlsbad, California, CA) was added to OPTI medium (Gibco, USA) containing si-Smad7. After being incubated for 20 min, the mixtures were added to AF cells. Si-NC was served as a negative control. The efficacies of transfection were determined by western blotting and real-time PCR.


## 2.5. Quantitative Real-Time-PCR

Total RNA was obtained from AF cells using an RNA isolation kit (Invitrogen) according to the manufacturer’s protocol. cDNA was generated by using the cDNA synthesis kit (Roche). A qRT-PCR method based on SYBR Green detection was performed for gene expression quantification using the primers. The primers were as follows: humanSMAD7 forward: 5′-CGGAAGTCAAGAGGCTGTGT3′, reverse: 5′-TGGACAGTCTGCAGTTGGTT-3′; Rat Smad7 forward: 5′-AGGCATTCCTCGGAAGTCAA-3′, reverse: 5′-TGGACAGTCTGCAGTTGGTTTG-3′. Real-time-PCR was performed with ABI StepOne Plus equipment. GAPDH was used as an internal control to normalize the expression levels of different genes. The relative complementary DNA ratio was calculated using the value of threshold cycles (Ct). The amplification efficiency between the target and the reference control GAPDH was compared by using the 2–ΔΔCt method.


## 2.6. Western-Blot Analysis

The AF cells were collected at 12, 24, and 48 h after recombinant adenovirus infection. The whole cell lysis was extracted using Cell Lysis Buffer for Western and IP (Beyotime, P0013) added with PMSF (1 : 100) [23]. The relative protein levels were calculated versus GAPDH. The primary antibodies used in this study were as follows: GAPDH, Bcl-2, Bax, Bcl-XL, Cyt-c, procaspase 9, cleaved-caspase 9, procaspase 3, cleaved-caspase 3, Smad7 (Proteintech, USA, 1 : 1000), Smad4 (Proteintech, USA, 1 : 1000), total-smad2 (Proteintech, USA, 1 : 1000), total-smad3 (Proteintech, USA, 1 : 1000), P-smad2 (CST, USA, 1 : 1000), and P-smad3 (CST, USA, 1 : 1000). Following incubation with a primary antibody, the membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit or anti-goat second antibody. The membranes were then washed thrice, and the immunoreactive bands were visualized using NBT/BCIP as a substrate. Densitometric analysis was done using the NIH ImageJ Software to quantify the protein present in the detected bands. GAPDH content was assayed as standardization of sample loading. Quantitative densitometric values of each protein of interest were normalized to GAPDH.


## 2.7. Nuclear Staining Using Hoechst 33258

Three groups of AF cells were seeded in 96-well culture plates and added with Hoechst 33258 dye solution (Beyotime, C0003) followed by removing the culture media at 12, 24, and 48 h, respectively. The procedures were done as previously described [21]. After washing with PBS twice, AF cells were added with 0.5 ml 4% formaldehyde solution and fixed at RT for 10 min. Then, AF cells were washed with PBS for 2-4 times and incubated with Hoechst 33258 solution for 20-60 min in the dark at RT. Cells were observed under a fluorescence microscope under UV light followed by washing with PBS and adding with antifade reagent.


## 2.8. Cell Apoptosis Assay Using Flow Cytometry

AF cells overexpressingSmad7 were first collected for apoptosis assay using flow cytometry at 12, 24, and 48 h, respectively. And then, we used LEHD-FMK to confirm the involvement of the mitochondria-mediated pathway in the apoptosis of rat AF cells after 48 h of Smad7 overexpression. The experiments were performed according to the manual of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen TM) [7]. In brief, AF cells were collected by centrifuge at 1000 g for 5 min followed by wash with PBS and digestion using Trypsin-EDTA. After resuspension in PBS, cell numbers were counted and 5-10×104 cells were aliquoted for staining with Annexin V-FITC solutions for 10 min in the dark at RT. Propidium iodide (PI) was then added to the AF cells. All the stained cells were analyzed using a flow cytometry platform for Annexin V-FITC and PI.


## 2.9. Suppression of Apoptosis by Z-LEHS-FMK

Z-LEHD-FMK (Caspase 9 inhibitor) was purchased from MedChemExpress (MCE), and the Cat. No. is HY-P1010.AF cells were seeded in 12-well culture plates at a density of5×105 cell/plate. All the plates were divided into four groups, with three plates each group. When the confluences of cells reached 80-90%, cell culture media were changed. The cells in one group were pretreated with 6 μl Z-LEHD-FMK for 2 h after being infected with Smad7 overexpression adenovirus. The other three groups were infected with Smad7 overexpression adenovirus, empty adenovirus, or nothing. Cells were collected after 48 h to test apoptosis rate.


## 2.10. Immunofluorescence Analysis

AF cells were seeded on sheet glasses in plates. AF cells were fixed by 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked by 5% BSA (bovine serum albumin) for 1 h. The AF cells were incubated with p-Smad3 (rabbit monoclone antibody, 1 : 200, Abcam) and p-Smad2 (rabbit monoclone antibody, 1 : 400, CST) primary antibodies overnight at 4°C. AF cells were washed with PBST thrice. Finally, AF cells were incubated with Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L). Photographs were taken by Olympus BX51.


## 2.11. Statistical Analysis

All data were analyzed using SPSS11.5 statistical software. Comparisons of data between groups were analyzed using one-way ANOVA, Kruskal-WallisH-test, or independent-samples t-test according to the distribution of data and homogeneity of variance. p values less than 0.05 were considered statistically significant.


## 3. Results

### 3.1.Smad7 Is Highly Expressed in the Human Degenerative Discs

The expression ofSmad7 in degenerative NP, EP, and AF was significantly higher than that in the nondegenerative counterpart (Figure 1(a)). The Smad7 protein expressions showed similar variation trends as compared with PCR analysis (Figure 1(b)). But Smad7 expressions were much higher in the AF than in NP and EP, which indicated that Smad7 might have a more important role in the AF than in the other components of the disc.Figure 1
The expression ofSmad7 in human degenerative and non-degenerative intervertebral discs. (a) qRT-PCR assays to detect the mRNA expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. (b) Western blotting assays to detect the protein expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. p values less than 0.05 were considered statistically significant. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate; ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times.
(a)(b)(c)


### 3.2.IL-1β Induces Apoptosis in Rat AF Cells through Its Regulations on Smad7

It has been proved thatIL-1β induces apoptosis in AF cells [18, 24, 25]. We investigated whether Smad7 was involved in the IL-1β-induced apoptosis of AF cells. With the stimulation of IL-1β, the apoptosis-related proteins, such as cleaved caspase-9 and cleaved caspase-3, significantly increased in the AF cells (Figure 2(a)). Smad7 mRNA (Figure 2(b)) and protein (Figure 2(c)) expressions were also upregulated with time in these AF cells.Figure 2
IL-1β increases the expression of Smad7 in rat AF cells along with apoptosis. (a) Western blotting assays to detect the protein expression of cleaved-caspase 9 and cleaved-caspase 3 in AF cells treated with IL-1β. (b) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with IL-1β for various times (0, 6, 12, 18, 24, and 48 h). (c) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with IL-1β for various times (0, 6, 12, 18, 24, and 48 h). p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times.
(a)(b)(c)Successful knockdown ofSmad7 in AF cells was verified at both the gene (Figure 3(a)) and protein (Figure 3(b)) levels. With the knockdown of Smad7 in AF cells, the IL-1β-induced apoptosis was greatly inhibited (Figure 3(c)). The expression of apoptosis-related proteins, such as Bax/Bcl-2, cleaved caspase-9, and cleaved caspase-3, was also significantly suppressed (Figure 3(d)).Figure 3
Smad7 Knockdown alleviates the IL-1β-induced apoptosis of AF cells. (a) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with si-NC, si-Smad7-1, si-Smad7-2, and si-Smad7-3. (b) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with si-NC, si-Smad7-1, si-Smad7-2, and si-Smad7-3. (c) Representative pictures of TUNEL-stained AF cells treated with si-NC, si-NC+IL-1β, and si-Smad7+IL-1β. (d) Western blotting assays to detect the protein expression of Bax, Bcl-2, cleaved-caspase 9, and cleaved-caspase 3 in AF cells treated with si-NC, IL-1β, and si-Smad7+IL-1β. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Scale bar: 10 μM.
(a)(b)(c)(d)Overexpression ofSmad7 in AF cells with adenovirus infection was also successful (Figures 4(a) and 4(b)). On the contrary, with the overexpression of Smad7 in AF cells, the IL-1β-induced apoptosis was greatly exaggerated (Figure 4(c)). More chromosome condensation and nuclear fragmentation under Hoechst 33258 staining were seen in the cells (Figure 4(d)). Taken together, these results indicated that IL-1β-induced apoptosis in rat AF cells through its regulations on Smad7.Figure 4
Overexpression ofSmad7 exaggerates the IL-1β-induced apoptosis (a) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with Con, Ad-Con and Ad-Smad7. (b) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with Con, Ad-Con, and Ad-Smad7. (c) Representative flow cytometry graphs of rat AF cells stained with Annexin V-FITC and PI. Gates were set as follows: Q1 (Annexin V-PI+), necrosis cells; Q2 (Annexin V+PI+), late-stage apoptotic cells; Q3 (Annexin V-PI-), live cells; Q4 (Annexin V+PI-), early-stage apoptotic cells. Apoptotic rates of three groups of rat AF cells at different time points. The percentages of Annexin V+ cells were calculated at Con, Ad-Con, and Ad-Smad7 at 12, 24, and 48 h, respectively. (d) Rat AF cells stained by Hoechst 33285 at 12, 24, and 48 h. Original magnification: ×200. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Con: control rat AF cells; Ad-Con: rat AF cells infected by adenovirus taking empty plasmid; Ad-Smad7: rat AF cells infected by adenovirus taking Smad7 overexpression plasmid. Original magnification: ×100. Scale bar: 10 μM.
(a)(b)(c)(d)


### 3.3.Smad7 Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway

There are two main apoptotic pathways: the extrinsic (also called death receptor) pathway and the intrinsic (also called mitochondrial) pathway [26]. The extrinsic pathway requires external stimulation, and this occurs via a death receptor (DR). The intrinsic pathway involves a wide array of stimuli that are sensed intracellularly, including cytokine deprivation, DNA damage, and endoplasmic reticulum (ER) stress [27]. These diverse apoptotic stresses converge to trigger one crucial event—mitochondrial outer membrane permeabilization (MOMP) [28]. It is reported that TGF-β1-mediated apoptosis was associated with Smad7-dependent mitochondrial Bcl-2 expression [29] and Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells [30]. These studies indicated that Smad7 might lead to cell apoptosis via the mitochondria signaling pathway.To investigate whetherSmad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria signaling pathway, we measured the protein expression of different biomarkers of the mitochondria-mediated apoptosis. Western blot analysis revealed that the protein level of Bax in rat AF cells overexpressing Smad7 significantly increased, while the levels of both Bcl-2 and Bcl-XL significantly decreased, which resulted in even greater increase in the ratio of Bax/Bcl-2 (Figure 5(a)). Meanwhile, the expression of both active cleaved caspase-3 and cleaved caspase-9 also increased significantly in the AF cells with Smad7 overexpression. However, the protein expressions of procaspase-3 and procaspase-9 did not show any significant changes (Figure 5(a)). In addition, the expression of Cyt-c also increased significantly (Figure 5(b)). These results indicated that the mitochondrial pathway is likely involved in the Smad7-mediated apoptosis induced by IL-1β. Smad7 may promote Bax expression by inhibiting Bcl-2 and Bcl-XL expression and activating the mitochondria-mediated apoptosis pathway.Figure 5
Smad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. (a) Western-blot analysis of Bax, Bcl-2, Bcl-XL, procaspase 9, cleaved-caspase 9, procaspase 3, and cleaved caspase3 in whole cell lysis from Con, Ad-Con and Ad-Smad7-infected rat AF cells. (b) Western-blot analysis of Cyt-c in whole cell lysis from Con, Ad-Con, and Ad-Smad7 infected rat AF cells. (c) Western blot analysis of procaspase 9 and active cleaved-caspase 9. (d) Representative flow cytometry graphs of rat AF cells stained with Annexin V-FITC and PI after treatment with Z-LEHS-FMK. (e) Immunofluorescence assays of cleaved-caspase 9 in AF cells. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Con, Ad-Con, Ad-Smad7-infected rat AF cells, and Ad-Smad7-infected rat AF cells treated with Z-LEHS-FMK. Scale bar: 20 μM.
(a)(b)(c)(d)(e)To further confirm the involvement of the mitochondria pathway in theSmad7-mediated apoptosis, AF cells with overexpression of Smad7 were treated with Z-LEHD-FMK, an inhibitor of caspase-9. After treatment with Z-LEHD-FMK, the protein expression of active cleaved-caspase-9 in Ad-Smad7-infected cells was downregulated by one-fold (Figure 5(c)), which is consistent with results reported in previous studies [31–33]. The apoptosis percentage was significantly lower in Ad-Smad7-infected cells treated with Z-LEHD-FMK than in cells without Z-LEHD-FMK treatment; however, it was still higher than that in Con and Ad-Con cells (Figure 5(c)). This suggested that Z-LEHD-FMK could not completely inhibit the Smad7-mediated apoptosis in AF cells. Immunofluorescence assay for the detection of protein expression of cleaved caspase-9 showed that Ad-Smad7 increased the level of cleaved caspase-9, while Z-LEHD-FMK weakened this effect of Ad-Smad7 (Figure 5(d)). These results further provided evidence that Smad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria signaling pathway.


### 3.4.Smad7 Regulates the IL-1β-Induced Apoptosis in AF Cells by Interfering with the Formation of Smad2/Smad3/Smad4 Complex

With the stimulation ofIL-1β, the protein expressions of p-Smad2, p-Smad3, and Smad4 significantly decreased in the AF cells (Figure 6(a)). However, in cells with a successful Smad7 knockdown, the expression of p-Smad2, p-Smad3, and Smad4 was not affected by IL-1β treatment. Furthermore, in the cells with Smad7 knockdown, the nuclear translocation of p-Smad2/p-Smad3 significantly increased (Figure 6(b)). These results demonstrated that Smad7 could regulate the IL-1β-induced apoptosis in AF cells by interfering with the formation of Smad2/Smad3/Smad4 complex.Figure 6
Smad7 regulates the IL-1β-induced apoptosis in AF cells by interfering with the formation of Smad2/Smad3/Smad4 complex. (a) Western blotting assays to detect the protein expression of Smad7, Smad4, p-Smad2, Smad2, p-Smad3, and Smad3 in AF cells treated with si-NC, IL-1β, and si-Smad7+IL-1β. (b) Immunofluorescence assays of p-Smad2 and p-Smad3 in AF cells treated with si-NC, IL-1β, si-Smad7, and si-Smad7+IL-1β. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Scale bar: 10 μM.
(a)(b)


## 3.1.Smad7 Is Highly Expressed in the Human Degenerative Discs

The expression ofSmad7 in degenerative NP, EP, and AF was significantly higher than that in the nondegenerative counterpart (Figure 1(a)). The Smad7 protein expressions showed similar variation trends as compared with PCR analysis (Figure 1(b)). But Smad7 expressions were much higher in the AF than in NP and EP, which indicated that Smad7 might have a more important role in the AF than in the other components of the disc.Figure 1
The expression ofSmad7 in human degenerative and non-degenerative intervertebral discs. (a) qRT-PCR assays to detect the mRNA expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. (b) Western blotting assays to detect the protein expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. p values less than 0.05 were considered statistically significant. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate; ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times.
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## 3.2.IL-1β Induces Apoptosis in Rat AF Cells through Its Regulations on Smad7

It has been proved thatIL-1β induces apoptosis in AF cells [18, 24, 25]. We investigated whether Smad7 was involved in the IL-1β-induced apoptosis of AF cells. With the stimulation of IL-1β, the apoptosis-related proteins, such as cleaved caspase-9 and cleaved caspase-3, significantly increased in the AF cells (Figure 2(a)). Smad7 mRNA (Figure 2(b)) and protein (Figure 2(c)) expressions were also upregulated with time in these AF cells.Figure 2
IL-1β increases the expression of Smad7 in rat AF cells along with apoptosis. (a) Western blotting assays to detect the protein expression of cleaved-caspase 9 and cleaved-caspase 3 in AF cells treated with IL-1β. (b) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with IL-1β for various times (0, 6, 12, 18, 24, and 48 h). (c) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with IL-1β for various times (0, 6, 12, 18, 24, and 48 h). p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times.
(a)(b)(c)Successful knockdown ofSmad7 in AF cells was verified at both the gene (Figure 3(a)) and protein (Figure 3(b)) levels. With the knockdown of Smad7 in AF cells, the IL-1β-induced apoptosis was greatly inhibited (Figure 3(c)). The expression of apoptosis-related proteins, such as Bax/Bcl-2, cleaved caspase-9, and cleaved caspase-3, was also significantly suppressed (Figure 3(d)).Figure 3
Smad7 Knockdown alleviates the IL-1β-induced apoptosis of AF cells. (a) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with si-NC, si-Smad7-1, si-Smad7-2, and si-Smad7-3. (b) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with si-NC, si-Smad7-1, si-Smad7-2, and si-Smad7-3. (c) Representative pictures of TUNEL-stained AF cells treated with si-NC, si-NC+IL-1β, and si-Smad7+IL-1β. (d) Western blotting assays to detect the protein expression of Bax, Bcl-2, cleaved-caspase 9, and cleaved-caspase 3 in AF cells treated with si-NC, IL-1β, and si-Smad7+IL-1β. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Scale bar: 10 μM.
(a)(b)(c)(d)Overexpression ofSmad7 in AF cells with adenovirus infection was also successful (Figures 4(a) and 4(b)). On the contrary, with the overexpression of Smad7 in AF cells, the IL-1β-induced apoptosis was greatly exaggerated (Figure 4(c)). More chromosome condensation and nuclear fragmentation under Hoechst 33258 staining were seen in the cells (Figure 4(d)). Taken together, these results indicated that IL-1β-induced apoptosis in rat AF cells through its regulations on Smad7.Figure 4
Overexpression ofSmad7 exaggerates the IL-1β-induced apoptosis (a) qRT-PCR assays to detect the mRNA expression of Smad7 in AF cells treated with Con, Ad-Con and Ad-Smad7. (b) Western blotting assays to detect the protein expression of Smad7 in AF cells treated with Con, Ad-Con, and Ad-Smad7. (c) Representative flow cytometry graphs of rat AF cells stained with Annexin V-FITC and PI. Gates were set as follows: Q1 (Annexin V-PI+), necrosis cells; Q2 (Annexin V+PI+), late-stage apoptotic cells; Q3 (Annexin V-PI-), live cells; Q4 (Annexin V+PI-), early-stage apoptotic cells. Apoptotic rates of three groups of rat AF cells at different time points. The percentages of Annexin V+ cells were calculated at Con, Ad-Con, and Ad-Smad7 at 12, 24, and 48 h, respectively. (d) Rat AF cells stained by Hoechst 33285 at 12, 24, and 48 h. Original magnification: ×200. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Con: control rat AF cells; Ad-Con: rat AF cells infected by adenovirus taking empty plasmid; Ad-Smad7: rat AF cells infected by adenovirus taking Smad7 overexpression plasmid. Original magnification: ×100. Scale bar: 10 μM.
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## 3.3.Smad7 Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway

There are two main apoptotic pathways: the extrinsic (also called death receptor) pathway and the intrinsic (also called mitochondrial) pathway [26]. The extrinsic pathway requires external stimulation, and this occurs via a death receptor (DR). The intrinsic pathway involves a wide array of stimuli that are sensed intracellularly, including cytokine deprivation, DNA damage, and endoplasmic reticulum (ER) stress [27]. These diverse apoptotic stresses converge to trigger one crucial event—mitochondrial outer membrane permeabilization (MOMP) [28]. It is reported that TGF-β1-mediated apoptosis was associated with Smad7-dependent mitochondrial Bcl-2 expression [29] and Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells [30]. These studies indicated that Smad7 might lead to cell apoptosis via the mitochondria signaling pathway.To investigate whetherSmad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria signaling pathway, we measured the protein expression of different biomarkers of the mitochondria-mediated apoptosis. Western blot analysis revealed that the protein level of Bax in rat AF cells overexpressing Smad7 significantly increased, while the levels of both Bcl-2 and Bcl-XL significantly decreased, which resulted in even greater increase in the ratio of Bax/Bcl-2 (Figure 5(a)). Meanwhile, the expression of both active cleaved caspase-3 and cleaved caspase-9 also increased significantly in the AF cells with Smad7 overexpression. However, the protein expressions of procaspase-3 and procaspase-9 did not show any significant changes (Figure 5(a)). In addition, the expression of Cyt-c also increased significantly (Figure 5(b)). These results indicated that the mitochondrial pathway is likely involved in the Smad7-mediated apoptosis induced by IL-1β. Smad7 may promote Bax expression by inhibiting Bcl-2 and Bcl-XL expression and activating the mitochondria-mediated apoptosis pathway.Figure 5
Smad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. (a) Western-blot analysis of Bax, Bcl-2, Bcl-XL, procaspase 9, cleaved-caspase 9, procaspase 3, and cleaved caspase3 in whole cell lysis from Con, Ad-Con and Ad-Smad7-infected rat AF cells. (b) Western-blot analysis of Cyt-c in whole cell lysis from Con, Ad-Con, and Ad-Smad7 infected rat AF cells. (c) Western blot analysis of procaspase 9 and active cleaved-caspase 9. (d) Representative flow cytometry graphs of rat AF cells stained with Annexin V-FITC and PI after treatment with Z-LEHS-FMK. (e) Immunofluorescence assays of cleaved-caspase 9 in AF cells. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Con, Ad-Con, Ad-Smad7-infected rat AF cells, and Ad-Smad7-infected rat AF cells treated with Z-LEHS-FMK. Scale bar: 20 μM.
(a)(b)(c)(d)(e)To further confirm the involvement of the mitochondria pathway in theSmad7-mediated apoptosis, AF cells with overexpression of Smad7 were treated with Z-LEHD-FMK, an inhibitor of caspase-9. After treatment with Z-LEHD-FMK, the protein expression of active cleaved-caspase-9 in Ad-Smad7-infected cells was downregulated by one-fold (Figure 5(c)), which is consistent with results reported in previous studies [31–33]. The apoptosis percentage was significantly lower in Ad-Smad7-infected cells treated with Z-LEHD-FMK than in cells without Z-LEHD-FMK treatment; however, it was still higher than that in Con and Ad-Con cells (Figure 5(c)). This suggested that Z-LEHD-FMK could not completely inhibit the Smad7-mediated apoptosis in AF cells. Immunofluorescence assay for the detection of protein expression of cleaved caspase-9 showed that Ad-Smad7 increased the level of cleaved caspase-9, while Z-LEHD-FMK weakened this effect of Ad-Smad7 (Figure 5(d)). These results further provided evidence that Smad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria signaling pathway.


## 3.4.Smad7 Regulates the IL-1β-Induced Apoptosis in AF Cells by Interfering with the Formation of Smad2/Smad3/Smad4 Complex

With the stimulation ofIL-1β, the protein expressions of p-Smad2, p-Smad3, and Smad4 significantly decreased in the AF cells (Figure 6(a)). However, in cells with a successful Smad7 knockdown, the expression of p-Smad2, p-Smad3, and Smad4 was not affected by IL-1β treatment. Furthermore, in the cells with Smad7 knockdown, the nuclear translocation of p-Smad2/p-Smad3 significantly increased (Figure 6(b)). These results demonstrated that Smad7 could regulate the IL-1β-induced apoptosis in AF cells by interfering with the formation of Smad2/Smad3/Smad4 complex.Figure 6
Smad7 regulates the IL-1β-induced apoptosis in AF cells by interfering with the formation of Smad2/Smad3/Smad4 complex. (a) Western blotting assays to detect the protein expression of Smad7, Smad4, p-Smad2, Smad2, p-Smad3, and Smad3 in AF cells treated with si-NC, IL-1β, and si-Smad7+IL-1β. (b) Immunofluorescence assays of p-Smad2 and p-Smad3 in AF cells treated with si-NC, IL-1β, si-Smad7, and si-Smad7+IL-1β. p values less than 0.05 were considered statistically significant. ∗∗∗p<0.001,∗∗p<0.01,and∗p<0.05. All experiments were performed at least three times. Scale bar: 10 μM.
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## 4. Discussion

Smad7 is a negative regulatory protein involved in the signal transduction of TGF-β pathway. It blocks signal transduction and inhibits the transcription of TGF-β gene. In our previous study, we observed that Smad7 was highly expressed in degenerative intervertebral discs of rats and that this was closely related to the catabolism and apoptosis of disc cells [13]. However, the underlying mechanisms remained unknown.A clue indicating a potential role ofSmad7 in IVDD came from the studies showing that TGF-β1 could promote proteoglycan synthesis and cell proliferation of NP and AF cells cultured in vitro and in vivo [7, 34–37]. As a downstream molecule of TGF-β1, exploring the role and mechanism of Smad7 in intervertebral disc degeneration has grabbed our interest. In this study, we found that Smad7 was highly expressed in human degenerative discs. Combined with the finding in our previous animal study that the expression of Smad7 significantly increased in the degenerative discs of rats, we verified that Smad7 should be involved in the pathophysiological process of intervertebral disc degeneration.It has been proved thatIL-1β has important roles in the pathogenesis of disc degeneration by stimulating the apoptosis in disc cells [17, 18]. In this study, we verified that IL-1β could stimulate apoptosis in the AF cells isolated and cultured from rats and found increased Smad7 expression in the cells that along with this IL-1β-induced apoptosis. In order to investigate the role of Smad7 in the apoptosis of AF cells and the underlying signaling pathway, Smad7 was overexpressed or knocked down in the AF cells under IL-1β stimulation. Overexpression of Smad7 in the AF cells exaggerated the IL-1β-induced apoptosis, while knockdown of Smad7 in the cells significantly diminished the IL-1β-induced apoptosis. Meanwhile, we found that with the increase of apoptosis in the cells with Smad7 overexpression, the proteins in the mitochondrial mediated apoptosis pathway, such as active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Smad7 regulated the apoptosis of the AF cells through the mitochondrial signaling pathway. Our results also demonstrated that Z-LEHD-FMK did not completely block the activation of caspase-9 in the AF cells; it did not completely offset the increased apoptosis in the AF cells with Smad7 overexpression. The reasons for this incompetency of Z-LEHD-FMK might be as follows: (1) Z-LEHD-FMK did not completely bind to caspase-9 in the AF cells, resulting in an incomplete inhibition of apoptosis in the cells with Smad7-overexpression. (2) Caspase-9 in the middle of the apoptotic cascade is the upstream molecule of caspases 3, 6, and 7 [28, 38, 39]. Smad7 activated the downstream molecules of caspase-9 through other means. (3) Smad7 did not completely activate the apoptosis through the mitochondrial pathway, and hence, other pathways might be involved.High expression ofSmad7 was reported in chondrocytes under IL-1β stimulation [19]. Although IL-1β is proved to be able to induce disc cell apoptosis and disc degeneration, the expression of Smad7 in the AF cells under IL-1β stimulation has never been described. In our study, we found that IL-1β led to an increase in Smad7 expression and a decrease in p-Smad2, p-Smad3, and Smad4 expressions in the AF cells. After successful Smad7 knockdown in the AF cells, stimulation of IL-1β did not change the expressions of p-Smad2, p-Smad3, and Smad4. Combined with previous data and studies [25, 40, 41], we believed that Smad7 should be involved in the apoptosis of AF cells by interfering with the formation of the Smad2/Smad3/Smad4 complex and regulate the apoptosis through the mitochondrial signaling pathway.In conclusion, our study demonstrated thatSmad7 was highly expressed in human degenerative intervertebral discs and that Smad7 regulated apoptosis of the AF cells mainly through the mitochondria signaling pathway. Smad7 might be a potential target for the prevention and treatment of IVDD.



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*Source: 2912276-2022-06-26.xml*