The expression of cytokine and its significance for the intervertebral disks of Kazakhs

Background Proinflammatory cytokine IL‐6 and anti‐inflammatory cytokine IL‐10 are expressed in herniated intervertebral disks of Kazakhs, but their significance is not yet understood. Methods Specimens of herniated lumbar disk were collected from 30 patients with single disk herniation during lumbar discectomy. As a control, 10 specimens were collected postmortem from the cadavers of individuals who did not die from spinal disease. The clinical symptoms in all cases were evaluated preoperation by the JOA index. The expressions of IL‐6 and IL‐10 in the degenerative disk cells of each specimen were detected using immunohistochemical staining. The amounts of IL‐6 and IL‐10 in each intervertebral disk were assessed by enzyme‐linked immunosorbent assay. Results The expression levels of IL‐6 and IL‐10 were significantly higher in the patient group than in the control group. Statistical analysis showed differences between ethnic Kazakh and Han patients in both the expression of IL‐6 and JOA scores. Immunohistochemical staining showed that in the patient group, IL‐6 was found in 28 patients and IL‐10 was found in 27 patients. Conclusion The high expression of cytokines, such as IL‐6 and IL‐10, has an important relationship with disk degeneration. Of course, age is another factor in cell apoptosis of degenerative disks. More research is necessary to discover how ethnicity and heredity impact the levels of expressed cytokines.

consisted of 16 men and 14 women, aged 23-71 years, with a mean age of 44.5 years. The individual intervertebral herniated disks obtained during lumbar discectomy consisted of the following: 2 of L3-L4, 15 of L4-L5, and 13 of L5-S1. All patients had a history of typical lower back pain, without acute or chronic infections, diabetes, etc., and their JOA scores were assessed preoperatively by the same surgeon. During surgery, it was confirmed that there were 25 cases of prominent type disk herniation and five cases of prolapse, with no evidence of any bulging cases among the total 30 cases. The control group consisted of lumbar intervertebral disk tissue extracted from the cadavers of 10 recently deceased individuals who had died from nonvertebral diseases. These individuals were six men and four women, aged 30-68 years, with a mean age of 46.6 years. Their cadavers provided specimens of L4-5 or L5-S1 nucleus pulpous and were checked during surgical tissue removal to have no evidence of intervertebral disk herniation.

| Experimental methods
After surgical removal of tissue from the body, all disk tissue from both the patient and control groups was washed repeatedly with phosphate-buffered saline (0.01% PBS). 4,[6][7][8][9] After this washing to remove any contamination, such as blood, each specimen was divided into two samples and placed in liquid nitrogen for preservation. After specimen collection was completed, the remainder of the experimental process was carried out by a laboratory technician.
For each of the 40 specimens, part of one sample was made into paraffin-embedded sections, used for HE staining, and the remaining sections of the paraffin block were used for immunohistochemistry.
The main methods and processes of immunohistochemistry were as follows: after cutting each of the paraffin-embedded tissue sections to 4-μm thick, they were placed in an oven at 60°C and baked for 2 hours. The samples were then put through xylene dewaxing and graded alcohol dehydration, placed in 3% H 2 O 2 for 10 minutes, and then under the microwave 0.01 mol/L citrate antigen retrieval solution repair for 10 minutes. Following this, the samples were placed in a solution of 0.01% PBS wash to which the primary antibody (IL-6 and IL-10 antibody) was added. After 1 hour shaking in a 37°C humidified incubator chamber, the samples were placed in a 0.01% PBS wash with added secondary antibody (universal IgG antibody-HRP polymer) and stored in a 4°C refrigerator overnight. The next day, the samples were exposed for 5 minutes to a 0.01% PBS rinse containing freshly prepared DAB color and VIS color to stop the reaction, then thoroughly washed with distilled water. They were washed again with hematoxylin, dried with neutral resin amines, and mounted. After this, PBS was used to replace the primary antibody to make it negative for microscopic examination.
The other samples from each of the 40 specimens were used to measure the IL-6, IL-10 proteins by enzyme-linked immunosorbent assay (ELISA) technique.
The main methods and processes were as follows: 40 specimens were weighed, 200 mg was removed from each for the test, cell lysate RIPA 2 mL and PMSF 20 μL were added, homogenized, centrifuged at 2800 cm/s 2 for 10 minutes at 4°C, supernatant was selected and used for uniform detection, and carried out according to the experimental procedure kit instructions. ELISA kits were purchased from Hysen-

| Statistical analysis
The t test was used to assess the difference in IL-6 and IL-10 levels for the control group and the patient group, as well as the IL-6 levels in the Kazak and Han patients. Multivariate analysis assessed the variance between patients' JOA scores.

| DISCUSSION
In recent years, with the development of imaging techniques, the degree of lumbar disk herniation patients with pressure on the nerve root causing clinical symptoms is not always consistent. 5,7,[9][10][11] Therefore, recent studies indicate that pressure on the nerve root not only produces more than just a simple mechanical problem but also a fairly complex pathophysiological process, which involves biochemical substances such as cytokines which may be an additional source of pain. 10,11 The role of IL-6 and other inflammatory cytokines in degenerative joint disease has been confirmed as a cause of joint damage and has a similar effect in causing inflammation in lumbar disk degeneration. Takahashi found IL-6-positive cells in herniated lumbar disk tissue, but lacked a control group for study. IL-10 is considered to be an  1,6,[12][13][14][15][16] Some even pointed out that the cause of 74% of disk degeneration can be explained by genetic factors, and the literature has confirmed a much higher level of incidence for Caucasians than for people of color. [14][15][16] In this study, using multivariate variance for analysis, we found that Kazakh patients with high expression levels of IL-6 and a high JOA score were statistically significant as compared with Han Chinese patients. This study confirms this trend of racial and genetic differences, as the Kazakhs are the descendants of two sources, Caucasian and Mongoloid, while Hans are essentially Mongoloid in descent. 16 Of course, other factors such as climate may have an influence on health. North of the Tianshan Mountains, Kazakhs live mainly above 40°′ north latitude, with winters as long as 5-6 months. There remains much room for further study as the differences between Kazakh and Han genetics, living environments, cultures, and many other factors are beyond the scope of this study. This study only scratches the surface of examining the role of ethnicity and genetic factors in intervertebral disk degeneration. Disk degeneration and herniation is a complex process, and its mechanism is unknown. Further elucidation of the role of cytokines in intervertebral disk degeneration will undoubtedly provide clues for the prevention and treatment of degenerative disk disease. 14,17

AUTHORS' CONTRIBUTIONS
Miao Xiaogang designed the study. Kaken Habaxi attended and performed all the operations for specimen collection, Quanshan Hou and Liping Zhang did the preoperation and postoperation tasks, and took JOA scores, followed up with patients, and performed analysis and interpretation of data.

We acknowledge the help of Professor Miao Xiaogang and Kaken
Habaxi in designing this article and attending the operations.
As no individual patient's identity is revealed by the photographs, verbal consent for publication was obtained from the patient or their relative, either telephonic or on a follow-up visit.