Expression and Mechanism of Interleukin 1 (IL-1), Interleukin 2 (IL-2), Interleukin 8 (IL-8), BMP, Fibroblast Growth Factor 1 (FGF1), and Insulin-Like Growth Factor (IGF-1) in Lumbar Disc Herniation.

BACKGROUND The expression and mechanism of IL-1, IL-2, IL-8, BMP, FGF1, and IGF-1 in Sprague-Dawley (SD) rats with lumbar disc herniation were investigated. MATERIAL AND METHODS Immunohistochemical methods were applied to identify IL-1, IL-2, IL-8, BMP, FGF1, and IGF-1. PI3K, AKT protein, and mRNA expression were detected and analyzed by Western blot analysis. We selected 30 healthy SD rats and divided them into 2 groups to construct an animal model that was validated by immediate CT scanning. Cartilage tissues from the lumbar disc herniation (experimental) group and control group were obtained and compared. RESULTS The expression of BMP was not significantly different between the control group and the experimental group (P>0.05). FGF1: There was no significant difference in the expression of FGF1 (P>0.05) between the control group and the experimental group. Compared with the control group, the expression of IGF-1 in the experimental group was significantly higher (P<0.05); the expression of IL-1 in the experimental group was significantly higher (P<0.05); and the expression of IL-2 in the experimental group was also significantly higher (P<0.05). There was no significant difference in IL-8 between the experimental group and the control group (P>0.05). The expression levels of PI3K and AKT protein and mRNA were significantly higher than those in healthy controls (P<0.05). CONCLUSIONS After lumbar disc herniation occurred, the IGF-1 was first activated; the PI3K/AKT signaling pathway was later activated, which resulted in the expression of IL-1 and IL-2 inflammation-related factors being increased.

Background

Intervertebral disc herniation is a very common disease in clinical practice [1-6]. Most of the symptoms can be relieved or cured by non-operative therapy. Currently, imaging studies have confirmed that disc herniation can be gradually reduced or eliminated [7,8], and this morphological change of the protruding tissue is one of the reasons for effective non-operative treatment [9]. Whether the disc herniation is natural absorption or reduction can be determined by easy absorption of the rupture-type intervertebral disc, such as the free type and the pierced longitudinal ligament type [10-13]. The autoimmunity reaction caused by the prominent nucleus pulposus tissue is one of the mechanisms for reabsorption after the disc herniation [14,15]. The present study constructed an animal model of intervertebral disc herniation to observe and investigate the expression and mechanism of IL-1, IL-2, IL-8, BMP, FGF1, and IGF-1 in Sprague-Dawley (SD) rats with lumbar disc herniation. The cartilage tissues from the fractured and healthy divisions were examined and estimated based on the related cell factors.

Material and Methods

Model establishment

We purchased 30 SPF grade male SD rats (age: 5–6 weeks, weight: 170–200 g) from the Animal Center of Zhejiang University School of Medicine: 15 rats were assigned to the healthy (control group) and 15 rats were assigned to the lumbar disc herniation (experiment). Cartilage tissues were obtained from rats in both groups through resection under general anaesthesia induced with Ketamine, which is a new intramuscular injection. The operation was performed at the Second Affiliated Hospital of Zhejiang University School of Medicine, in which the lumbar disc herniation was exposed by a side incision. The cartilage of the lumbar disc herniation was exposed by separation and the same approach was applied for the healthy group.

HE staining

Paraffin-embedded sections were treated with xylene for 20 min, and then were treated again for another 20 min with fresh xylene. Infiltration in absolute alcohol for 7 min was followed by infiltration in 88% ethanol for 3 min. Sections were placed into distilled water for 3 min before soaking it in 75% ethanol for 3 min. Sections were stained with eosin for 3 min, then the remaining staining solution was washed away with tap water. We stained sections again with hematoxylin for 20 min, washed away the remaining staining solution with tap water, and put it in 75% ethanol for 3 min, 95% ethanol for 5 min, and absolute alcohol for 10 min. Finally, sections were deparaffined with fresh xylene for 20 min.

Immunohistochemistry test

After deparaffinization, sections were heated at medium heat for 2 min and high heat, for 2 min for antigen repair. After blocking for 5 h in 35°C antibody blocking solution and diluting the antibody with antibody blocking solution, sections were incubated with antibody overnight at 5°C. We washed the paraffin with PBS 3 times for 10 min each, diluted the second antibody with the antibody blocking solution, and incubated it at 35°C for 3 h. DAB working fluid was configurated to avoid response for 40–80 min, followed by washing the remaining DAB working fluid with tap water. For microscopic examination of cells, sections were re-stained with hematoxylin, dehydrated transparently, and examined on a neutral resin sheet.

Western blot test

We assessed the PI3K and AKT protein expression standard using the agent provided by Sigma and the PVDF (Tianjin Kameide Biotech). The related antibody, the RIPA cracking liquid, as well as the related chemiluminescence test kit, were all purchased from Synbio Technologies, Jiyun Biotechnology Company, and Beijing Protein Innovation, respectively.

RT-PCR experiment

AKT Sequence:

F: 5′-GGCGCACATCTACAGGATCAC-3′

R: 5′-CTATGCTCGACCCGACTGCA-3′

PI3K Sequence:

F: 5′-ACGATTCGCAGCTGAGTAATCACA-3′

R: 5′-ACTCCAAAGAGACTGGATCGAGATA-3′

Statistical analysis

Statistical analysis was performed using SPSS software, version 20.0. All data were analyzed using ANOVA and the independent t test was used to compare the 2 groups. P<0.05 indicates that the value is statistically significant.

Results

Control group: HE staining presented a light pink color for the normal cartilage, with the color even and the cartilage cell blue. The cartilage cells were arranged orderly into upper, middle, columnar, and cartilage layers.

Experiment group: HE staining showed the cell arrangement was disordered, with the cartilage matrix light in color and the cell surface loose.

The images of control and experiment groups are shown in Figure 1.

Figure 1

Images of control (A) and experiment (B) groups.

Immunohistochemistry

The images obtained from immunohistochemistry are shown in Figure 2. The brown granules indicate positive staining and blue indicates the nucleus. The joint crystal cells of both the experiment and the control groups were in average distribution with no clear cluster phenomenon. Comparison of the 2 groups showed no significant difference in the BMP expression (P<0.05). There was no significant difference in the expression of FGF1 between the 2 groups (P>0.05). The IGF-1 expression of the test group was higher than in the control group (P<0.05). The IL-1 expression of the experiment group was higher than in the control group (P<0.05). IL-6 expression in the experiment group was higher than in the control group (P<0.05). There was no significant difference in IL-8 expression between the experiment group and the control group (P>0.05).

Figure 2

Images obtained from immunohistochemistry (A, C, E, G, I, K – control group, B, D, F, H, J, L – experimental group) for A, B – BMP, C, D – FGF1, E, F – IGF-1, G, H – L-1, I, J – IL-2, and K, L – IL-8.

PI3K-AKT signal track protein and mRNA expression standard

The PI3K, AKT protein, and mRNA expression levels of the experiment group were significantly higher than those of the control group (P<0.05). PI3K protein and mRNA expression and AKT protein and mRNA expression obtained from the 2 groups are shown in Figures 3 and 4, respectively.

Figure 3

Comparison of PI3K protein and mRNA expression of the 2 groups.

Figure 4

Comparison of AKT protein and mRNA expression of the 2 groups.

Discussion

Insulin-like growth factor 1 (IGF-1) has an important role in promoting cell proliferation and apoptosis inhibition. However, previous studies in this area have mostly focused on malignant tumors [3,6,8,10,16], and there have been few studies on lumbar disc herniation. Relevant investigations proved that activation of the IGF-1 factor can activate the PI3K/AKT signal pathway [5,7-9]. Studies also suggest that the PI3K/AKT signaling pathway and interleukin have a positive role [10,14]. Therefore, we inferred that after lumbar disc herniation occurred, the body-mediated immune proliferative reaction may have a close correlation with this pathway.

Leukocyte interleukin 6 (IL-6) is a pleiotropic proinflammatory cytokine with many biological activities, including those that mediate inflammation and immune response. Studies have shown that IL-6 can inhibit the differentiation of bone marrow mesenchymal stem cells into chondrocytes by activating the MAPK/ERK signaling pathway and by osteogenic differentiation [11,12,17]. The lumbar intervertebral disc is the largest tissue without blood supply in the human body. The nucleus is encapsulated by the fibrous ring and is isolated from the blood circulation. The nutrition is mainly supplied by the diffusion of the cartilage endplate, so this tissue has its own immunity. After the breakthrough of the posterior longitudinal ligament type disc herniation, the longitudinal ligament of the protrusion enters the epidural and the tissue of the intervertebral disc exposed in the blood circulation to become antigen, resulting in autoimmune reaction to produce auto-antibodies or self-sensitized lymphocytes, which dissolve the protruding intervertebral disc. However, IL-1 and IL-6 are significantly increased in this process, which is consistent with results of the present study.

Interleukin 1 (IL-1) regulates immune reaction and inflammation [1,3,14], and expression of IL-1 in a variety of tissue factors is promoted by the synthesis of IL-2, IL-6, and IL-8 related cytokines. Expression of IL-1 in wet arthritis induces cartilage cells surrounding matrix degradation, resulting in cartilage cell apoptosis [18]. In the present study of the process of lumbar disc herniation, the mechanism of cartilage cells apoptosis was found to be the most important process. Through detection of related cytokines, this study found that the IL-1 and IL-6 expression levels in the cartilage of the experiment group were significantly higher than those in the healthy control group. This confirmed that the main expression of cytokines was IL-1 and IL-6 due to the lumbar disc herniation within the condyle DCS capsule/pouch and the body’s immune reaction. Thus, we suggest that the increased IL-1 and IL-6 expression levels can be attributed to the activation of the PI3K/AKT signal pathway.

Phosphoinositide 3 kinase/protein kinase B (PI3K/AKT) signal pathway is involved in abnormal proliferation of malignant tumors, and is also very important in cell metabolism, cell cycle control, and formation of blood vessels [5-7,10]. Therefore, we suggest that when lumbar disc herniation occurs, this pathway may be activated to repair the damaged cells and tissues. We found that the protein expression level and the mRNA expression level in the experiment group were significantly higher than in the control group. When we assessed the effect of intervertebral disk herniation on the expression of PI3K/AKT, there were 2 key proteins in this pathway in the experimental group, but no such association was found in healthy tissues and mRNA expression. The results confirmed that there was a clear association between increased expression of IL-1 and IL-6 and activation of the PI3K/AKT signal pathway. The related factors were measured to determine the activation potential of PI3K/AKT signal pathways. Some related studies have found that IGF-1 activates the PI3K/AKT signal pathway, promotes cell proliferation, and inhibits apoptosis. Our study shows that IGF-1 expression levels in the damaged experiment group were significantly higher than those in the control group.

Conclusions

After lumbar disc herniation occurs, IGF-1 is first activated, and then the downstream PI3K/AKT signaling pathway is activated, enhancing the related inflammation factors and IL-1 and IL-6 expression.