The meta-analysis conformed to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidelines, and registered in PROSPERO International Prospective Register of Systematic Reviews (CRD42019124118). This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (2018-KY-NSFC-025). Written informed consent was obtained from all the participants involved in this study. All procedures were performed in accordance with the guidelines the institutional research committees and with the Declaration of Helsinki.
Search strategy
Four electronic databases, including PubMed, Embase, Web of Science and China National Knowledge Infrastructure (CNKI), were searched by two reviewers (HJ and QX). The following keyword search string was used to identify studies: (IL-1 OR IL 1 OR interleukin-1 OR interleukin 1 OR IL-1RN) AND (polymorphism OR variant OR mutation OR SNPs) AND (disc degeneration OR disc disease OR low back pain). Additional studies were identified through a hand search of references listed in the reports and reviews. No language or publication date restrictions were used. The final literature search was conducted on February 5, 2020.
Inclusion and exclusion criteria
Eligible studies were included in meta-analysis according to the following inclusion criteria: (1) case-control design; (2) LDD diagnosed on the basis of clinical or/and radiologic examinations; (3) the study evaluated the association between IL-1α rs1800587 or IL-1β rs1143634 or IL-1RN (VNTR) polymorphisms and LDD risk; (4) genotype of control group conformed to the Hardy-Weinberg balance; and (5) sufficient data were provided to calculate the odds ratios (ORs) and 95% confidence intervals (CI). The exclusion criteria were as follows: (1) comments, reviews, or meta-analysis; (2) repeated publications; (3) study without available data. Based on the inclusion and exclusion criteria, eligible studies were identified independently by two reviewers (HJ and QX). The disagreements were resolved by a third reviewer (QW).
Data extraction
According to a standardized form, two investigators (HJ and QX) independently extracted data on outcomes for each study. The following information were collected from the included studies: (1) first author; (2) publication year; (3) country of enrollment; (4) ethnicity; (5) study design; (6) numbers of cases and controls; (7) characteristics of participants (gender and age); (8) diagnostic criteria; (9) source of control group; (10) allele or/and genotype frequencies; (11) P value for Hardy-Weinberg equilibrium (HWE) of control.
Methodological quality assessment
Methodological quality of studies was assessed using Critical Appraisal Skills Programme (CASP) [26]. For each question, there were three answers including “no”, “can’t tell” and “yes”, which respectively indicate scored 0, scored 1 and scored 2. The quality score ranged from 0 to 20, and a study with scored 15-20 represented a high-quality study.
Study population
Based on our previous study [27], we collected degenerative disc tissues (n=34) and normal disc tissues (n=21) from the LDD patients and the control subjects, respectively (Table S1). These disc samples were used to evaluate gene expressions via qRT-PCR and IHC. LDD patients were diagnosed as lumbar disc herniation by physical examination and MRI scan. The final diagnosis was verified by histopathology. The control subjects were the patients with traumatic lumbar vertebral fracture, who had no history of low back pain. According to Schneiderman’s classification [28], MRI evaluation of the control subjects showed no significant disc damage and degeneration before surgery (Schneiderman’s classification, Grade 1: 19 cases; Grade 2: 2 cases).
RNA extraction and qRT-PCR analysis
Nucleus pulposus tissues harvested from 55 subjects (34 cases and 21 controls) were lysed in TRIzol® (Invitrogen Inc, Carlsbad, CA, USA) and total RNA was extracted using an RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The reverse transcriptions (RT) were performed using PrimeScript RT Master Mix kit (Takara, Japan), with 1 μg total RNA used for the synthesis of the complementary DNA (cDNA) via using iScripts cDNA Synthesis kit (Quanta Biosciences, MD, USA). SYBR Green real-time PCR kit (Quanta Biosciences, MD, USA) was used to measure the relative mRNA levels, and samples normalized for GAPDH expression. All reactions were run on a real-time PCR system (Applied Biosystems) and analyzed using the comparative Ct (ΔΔCt) method (2-ΔΔCt with logarithm transformation). For profiling gene expressions, qRT-PCR was performed, using the primer pairs for IL-1α (5’-CCTCACCTTCCAGGAGAATGTG- 3’ and 5’-GCATCGCCCAGATTTTGTAG TG-3’), IL-1β (5’-CTGTCCTGCGTGTT GAAAGAT-3’ and 5’-TTCTGCTTGAGAG GTGCTGAT-3’), IL-1RN (5’-TTGTCCT GCTTTCTGTTCTCG-3’ and 5’-CTGTCC TGTGTCAAGTCTGG-3’), and GAPDH (5’-GACATGCCGCCTGGAGAAAC-3’ and 5’-AGCCCAGGATGCCCTTTAGT- 3’).
Immunohistochemical assay
Nucleus pulposus tissues were obtained from case and control groups (34 cases and 21 controls). Immunohistochemical assay was performed using a standard protocol as previously reported[27]. The sections were treated with 1/200 IL-1α antibody (ab7632, Cambridge, MA, USA), 1/200 IL-1β antibody (#2022, Cell Signaling, Danvers, MA, USA), 1/400 IL-1RN antibody (ab123235, Abcam, Cambridge, MA, USA) overnight at 4 °C and incubated with 1/400 secondary biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by treatment with VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). Based on stained slides, the number of positive cells was manually counted using Olympus BX43 upright microscope.
Statistical analysis
The association between IL-1α rs1800587, IL-1β rs1143634 and IL-1RN (VNTR) gene polymorphisms and LDD were estimated by odds ratio (OR) and 95% confidence intervals (CI). We evaluated the pooled ORs in five different genetic models. The heterogeneity of statistics was calculated by chi-square-based Q statistics and I2 statistics. Heterogeneity was considered to be effective, when P was less than 0.10 and I2 was greater than 50%. If there was significant heterogeneity (I2>50%), the random effect model was used. Otherwise, the fixed effect model was applied. Subgroup analysis of ethnicity was conducted to identify the source of heterogeneity. HWE was detected in control groups using the chi-square test. We performed the sensitivity test to assess the possible influence of one study on the pooled OR. In sensitivity test, studies were removed, in turn, from the overall analysis. Publication bias was tested by Begg’s funnel plot and Egger’s test. The qRT-PCR and IHC assay were calculated by the mean±standard error. Statistical difference between two groups was evaluated using unpaired Student t test. P<0.05 was considered to be statistically significant. Statistical analysis was performed by STATA 12.0 software (Stata, College Station, TX) and Revman 5.31 software (Nordic Cochrane Centre, Cochrane Collaboration, Copenhagen, Denmark).