Introduction
Diabetic neuropathic pain (DNP), one of the chronic complications of diabetes, is difficult to treat and results in a huge economic and psychological burden on patients [
1,
2]. Although an increasing number of studies are focusing on DNP, its underlying mechanisms appear to be complex and not yet completely understood.
Inflammasomes are complexes comprising a sensor protein, an adaptor protein, and an effector protein pro-caspase-1 [
3,
4]. Inflammasomes are categorized into two families—the nucleotide-binding domain, leucine-rich repeat-containing proteins (NLRs) [
3,
4], and the absence in melanoma 2 (AIM2)-like receptors (ALRs) [
5]. Once activated, its effector protein pro-caspase-1 produces cleaved-caspase-1, and subsequently cleaves pro-IL-1β to IL-1β, which then induces abnormal pain [
6]. NLRP3 is a key molecule within the inflammasome, which can be activated by thioredoxin-interacting protein (TXNIP) and has attracted much attention in the field of pain [
7‐
9]. Upregulation of NLRP3 inflammasomes of the sciatic nerve and dorsal root ganglion (DRG) is reportedly involved in DNP [
10,
11]. However, the epigenetic regulatory mechanisms underlying the modulation of NLRP3 upregulation, remain to be elucidated.
Epigenetic regulation includes the modifications of DNA, histones, and non-coding RNAs. Recent research has confirmed that epigenetic regulation contributes to DNP [
12‐
14]. DNA modification mainly includes DNA methylation and demethylation, both of which play vital roles in the regulation of gene expression. In mammals, DNA methylation is mainly mediated by the enzymes belonging to the DNA methyltransferase (DNMT) family, including DNMT1, DNMT3a, and DNMT3b, which transfer a methyl group to the C5 position of DNA cytosine to form 5-methylcytosine (5mC) [
15]. In general, DNA methylation suppresses gene expression. Methylated DNA is demethylated by ten-eleven translocation methylcytosine dioxygenases (TETs), which are capable of oxidizing 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally, 5-carboxycytosine (5caC), usually contributing to gene overexpression. TETs have three subtypes—TET1, TET2, and TET3 [
16]. Our previous study indicated that the methylation level of the genomic DNA promoter region was altered in STZ-diabetic mice DRG [
17]. Although it has already been validated that DNA methylation is involved in DNP, the focus of most research has been on DNMT-mediated DNA methylation [
13], while the role of TETs in DNP had not been reported to date.
Using high levels of glucose to treat cultivate human mesangial cells (HMCs) is an often-used in vitro hyperglycemia cell model. Researchers observed a significant increase in levels of mRNA and protein of TET2, while the expressions of TET1 and TET3 exhibited no change [
18]. Another study reported that using high levels of glucose to cultivate human umbilical vein endothelial cells (HUVECs), which were often used to study the effect of hyperglycemia on vascular endothelial in vitro, significantly increased the expression of NLRP3 [
19]. These findings indicate that high glucose (HG) is capable of upregulating both TET2 and NLRP3. Moreover, it is reported that inflammasome genes undergo DNA demethylation during monocyte-to-macrophage differentiation and that
TET2-siRNA inhibits this phenomenon [
20]. These findings suggest that TETs-mediated DNA demethylation may influence the expression of inflammasome genes. In this context, the present study aimed to investigate whether TETs mediated the upregulation of NLRP3 inflammasome of DRG involved in DNP and its underlying mechanisms.
Materials and methods
Animals
Four hundred and fifty healthy wild-type male and 60 female C57BL/6 mice (age: 6–8 weeks) were procured from Charles River Laboratories. The animals were housed in cages (4–5 animals per cage) maintained at a temperature of 23 ± 2 °C and under 12-h light/12-h dark photoperiod and were fed with ordinary chow. Experiments were performed in accordance with followed ARRIVE and RIGOR guidelines [
21]. All procedures were approved by the Experimental Animal Ethics Committee of Capital Medical University. Anesthesia and euthanasia of animals were consulted with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020).
Type 1 diabetes mouse model
In order to establish the type 1 diabetes mouse models, the mice were subjected to overnight fasting, following which they were administered intraperitoneal (
i.p.) injections of freshly prepared streptozotocin (STZ, 50 mg/kg/day, Sigma 0130) for five consecutive days. The control group mice were administered citrate buffer (5 mL/kg/day) for 5 days [
22]. Fasting blood glucose levels were measured from the tail vein using the ACCU-CHEK
® Mobile blood glucose meter (Roche). If the fasting blood glucose level was above 11.1 mmol/L, the mouse was considered diabetic and used for subsequent analyses. The body weights of the mice were also recorded.
Glucose tolerance test (GTT)
GTT was performed on non-fasted mice. Briefly, food was removed for 1 h, then mice were injected with 200 mg/mL of D-glucose (2 g/kg body weight,
i.p.). Blood glucose levels were tested at 0, 15, 30, 45, 60, 90, 120 min post-glucose administration according to the previous report [
23].
HbA1c test
HbA1c was tested by a glycosylated hemoglobin assay kit (A056-1-1, Nanjing Jiancheng Bioengineering Institute) [
24]. Briefly, blood of about 0.5–1 mL was harvested from the retro-orbital plexus in anticoagulant tubes, then centrifuged, and removed the supernatant. The sediment was washed in normal saline to obtain red blood cells solution. Double-distilled water was added to the above solution and mixed on a Vortex for 1 min to obtain dissolving blood. The absorbance of the dissolving blood was measured at 540 nm wavelength and the concentration of Hb was calculated according to the instruction of the kit. Acidified and hydrolyzed the dissolving blood and measured the absorbance at 443 nm. At last, the concentration of HbA1c was represented by the absorbance of every 10 g Hb (OD/10gHb). IFCC-HbA1c (%) = (OD/10gHb) × 0.001 + 0.0154; IFCC-HbA1c(mmol/mol) = 13 × IFCC-HbA1c (%) × 100–7.4.
Intraepidermal nerve fiber density
Intraepidermal nerve fiber density (INFD) was assessed as described previously with minor modification [
25]. Three randomly chosen 5-µm sections from each mouse were deparaffinized in xylene (1330-20-7, Shanghai Lingfeng Chemical Reagent, China), hydrated in decreasing concentrations of ethanol (100–75%), and rinsed in water. After antigen retrieval with citric acid (G1202, Servicebio, China) and endogenous peroxidase activity blocking with 3% H
2O
2 acid (Disinfection Technology, China), nonspecific binding was blocked by 10% goat serum (G1209, Servicebio) containing 3% BSA (G5001, Servicebio) for 30 min at room temperature. Then, PSD9.5 antibody (GB11277, ServiceBio, China) was added to the samples and incubated overnight at 4 °C. Slices were washed and incubated in secondary HRP-labeled antibodies (GB23303, ServiceBio) for 50 min at room temperature. Then, DAB chromogenic reaction was carried out with the commercial kit (G1211, Servicebio). Next, sections were counterstained with hematoxylin (G1340, Servicebio), dehydrated, and mounted in SweSuper clean Biomount medium (G1404, Servicebio). Intraepidermal nerve fiber profiles were counted blindly by independent investigators under a Nikon E100 microscope with a DS-U3 imaging system (Japan).
Behavioral test
The mechanical threshold of hind paws was detected using calibrated von Frey hairs (Stoelting, Wood Dale, IL, USA) as described in our previous research works [
17,
26,
27]. Briefly, the mice were placed on an elevated metal mesh floor and enclosed within a transparent plastic cage. The mice were allowed to habituate in this environment for a minimum of 30 min each day for 3 days prior to the test. Eight von Frey hairs (0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, and 1.4 g) were selected. Each trial commenced with the 0.16 g von Frey hair and was delivered perpendicularly to the central plantar surface of either of the hind paws for 3–5 s. The 50% paw withdrawal threshold (PWT) was measured using the “up-and-down” method and the following formula [
28]: 50% PWT(g) = 10
X + kd/10
4, where
X denotes the value of the final von Frey hair used,
k denotes the value from the pattern of positive/negative responses, and
d is the average increment (in log units) among the von Frey hairs used.
Thermal sensitivity was measured using the Hargreaves method. Briefly, the mouse was allowed to adapt to the environment within a ventilated plexiglass cage with a glass floor. When the mouse was awake, and in a calm state, a radiant heat (15 W power) was applied to the center of either of the hind paws. The paw withdrawal latency (PWL) was recorded three times, and the average was used as the final threshold value. There was a minimum of 5 min interval between each set of consecutive measurements. A time threshold of 20 s was set to prevent possible tissue damage.
Culture of mice DRG cells
DRG cells were isolated from naïve mice aged 4 weeks. Briefly, mice were anesthetized by isoflurane and then sacrificed quickly. The vertebral columns were removed and placed in an ice-cold DMEM solution (Gibco, C11995500BT). Then, about 150 DRGs were removed and pooled from 5 mice. DRGs were digested in 3% collagenase (Sigma, C9891) for 50 min and in 0.25% trypsin (Invitrogen, 15050065) for 10 min at 37 °C. The isolated DRG cells were seeded into poly-
d-lysine (Sigma, P0899) coated 35-mm dishes and cultured in culturing medium [DMEM medium (Gibco, 11966025) supplemented with 10% FBS (Vistech, SE100-B), 5 mM glucose (Sigma, G7021), and 0.6 nM insulin (Wanbang Biopharma, 41-532K)] at 37 °C in a water-saturated atmosphere with 5% CO
2 [
29]. After 3 days of culturing, neurons were incubated in a different medium for an additional 24 h. Control group: the same with culturing medium. Mannitol (MT) group: DMEM medium supplemented with 10% FBS, 5 mM glucose, 0.6 nM insulin, and 20 mM MT (Energy Chemical, E100520). High glucose (HG) group: DMEM medium supplemented with 10% FBS and 25 mM glucose. The medium was changed every 12 h.
In another set of experiments, after isolating and culturing DRG neurons for three days in Neurobasal media (Gibco, 21103049) plus 2% B-27 supplement (Gibco, 17504044), DGR cells were cultured with the following solution, respectively, for 24 h: Neurobasal media (Gibco, 21103049) containing 25 mM glucose was used as the control group, and Neurobasal media with additional 20 mM MT were used as another control. In order to mimic hyperglycemia in vitro, Neurobasal with additional 20 mM glucose (total 45 mM) was used in the HG group. And the B-27 supplement was switched to the B-27 supplement minus insulin (Gibco, A1895601) in the HG group.
RT-qPCR analysis
RT-qPCR assay was conducted as described before [
30]. Total mRNA was extracted from cells or tissues using TRIzol reagent (15596026, life technologies, USA) from DRGs or primary DRG neuronal cultures. The cDNA was synthesized using a reverse transcription kit (R223-01, Vazyme, China), and qPCR experiments (7500, Applied Biosystem) were performed according to the instructions. After the reaction was completed, the sample was stored at 4 °C, and the resulting threshold cycle (CT) value was the number of cycles required when the fluorescence signal reached a set threshold. The relative expression amount of each pair of samples was calculated from (2
−ΔΔCT).
GAPDH | TGTTCCTACCCCCAATGTGT | TGTGAGGGAGATGCTCAGTG |
TET2 | ATCCTTGCATTGGAGGGGTG | TTCCGGTCGGGATCGTTTAC |
TXNIP | ACGTGTGTCAGTCTCTGCTC | AGTGTGTCGGGCCACAATAG |
NLRP3 | CCAGCCAGAGTGGAATGACA | ACAAATGGAGATGCGGGAGA |
Caspase-1: | GGGACCCTCAAGTTTTGCC | GACGTGTACGAGTGGTTGTATT |
IL-1β | AACTCAACTGTGAAATGCCACC | CATCAGGACAGCCCAGGTC |
Western blot analysis
Western blots were performed as previous reported [
31]. Briefly, mice were euthanized with isoflurane anesthesia followed by rapid decapitation. The L3–L5 DRGs of the control and STZ-diabetic mice were retrieved (left and right sides, and a total of six DRGs from one mouse were pooled together) and subsequently disrupted using the RIPA lysis buffer. Briefly, the DRGs were sonicated and left on ice for 1 h, followed by centrifugation at 12,000
g for 15 min at 4 °C to obtain the supernatant. An aliquot of 50 μg of protein was mixed with loading buffer and then placed in boiling water for 5 min. The proteins were separated on the SDS-PAGE gel and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% non-fat milk dissolved in TBST for 1 h at room temperature and subsequently incubated overnight with the following antibodies at 4 °C: rabbit anti-TET1 (1:1000, GeneTex), rabbit anti-TET2 (1:1000, Millipore), rabbit anti-TET3 (1:1000, ABclonal), rabbit anti-TXNIP (1:1000, Santa), rabbit anti-NLRP3 (1:1000), rabbit anti-caspase-1 (1:1000, ABclonal), rabbit anti-IL-1β (1:1000, ABclonal), and mouse anti-α-tubulin (1:10,000, Biodragon). Next, the membrane was washed 3 times with TBST (for 5 min each time) and then incubated with the HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies. All antibodies had been diluted in 5% non-fat milk. Finally, the membrane was washed and exposed to ECL reagents for the detection of protein bands. The protein bands were scanned and analyzed using the Image J software.
Immunofluorescence staining
As reported before [
32], the mice were anesthetized with 1% pentobarbital sodium and then perfused with normal saline followed by a solution of 4% paraformaldehyde (PFA). The DRGs were post-fixed in 4% PFA for 4 h and then dehydrated sequentially in 20% and 30% sugar solutions. Each dehydrated DRG sample was excised into 10-µm-thick serial sections, which were then mounted on glass slides. After antigen retrieval and blocking with 5% fetal bovine serum, the DRG sections were co-incubated overnight at 4 °C with rabbit anti-TET2 antibody and DAPI with one of the following antibodies: mouse anti-NeuN (1:500, Sigma), mouse anti-NF200 (1:500, Sigma), or mouse anti-CGRP (1:500, Abcam). Subsequently, after washing the slides three times with phosphate buffered saline (PBS) (for 5 min each time), the sections were incubated with a mixture of Alexa Fluor 488 goat anti-rabbit IgG (H + L) and Alexa Fluor 568 goat anti-mouse IgG (H + L) (1:200, Invitrogen, Life technologies™, USA) 1 h in dark condition. Images were captured under a fluorescence microscope. We acquired at least 9 locations in the DRG per mouse: we quantified 3 images per section and 3 sections per mouse. We quantified at least 3 mice per experiment blindly.
DNA dot-blot assay
The levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) were determined using DNA dot-blot assays [
33]. Briefly, genomic DNA was extracted from the DRGs of mice according to the instructions of the kit (Solarbio, D1700). The extracted genomic DNA samples were categorized into three groups—62.5 ng, 125 ng, and 250 ng—based on their size and were then spotted on a nitrocellulose membrane using the Bio-dot microfiltration apparatus. The spotted membrane was placed in the ultraviolet crosslink equipment for 1 min and then at room temperature for 1 h. Subsequently, the membrane was blocked with 5% non-fat milk in TBST for 1 h, followed by incubation overnight at 4 °C with rabbit anti-5mC (1:1000, Merck), rabbit anti-5hmC (1:5000, Active Motif), rabbit anti-5fC (1:5000, Active Motif), and rabbit anti-5caC (1:5000, Active Motif) in TBST buffer with 5% non-fat milk. After washing the membrane three times with TBST (for 5 min each time), it was incubated with goat anti-rabbit secondary antibody (1:1000, HRP labeled) dissolved in 5% non-fat milk at room temperature for 1 h. The membrane was washed again three times (for 5 min each time) and exposed to the ECL reagent for detection. The intensity of the dot-blot image was analyzed using the Image J software. Meanwhile, the control DNA dot-blot membrane spotted as the same before was placed in 0.02% methylene blue diluted in 0.3 M sodium acetate to stain the DNA.
Intrathecal injection, intraperitoneal injection, and in vitro siRNA transfection
The mice (10–12 weeks) were anesthetized using isoflurane and then placed in a prone position. A lumbar puncture was performed using a 30-gauge needle connected to a 25-µL Hamilton microsyringe as reported before [
31]. The NLRP3 inhibitor MCC950 (InvivoGen, inh-mcc) and the siRNA (Santa Cruz, TET2-siRNA: sc-154205; Scr-siRNA: sc-44238) were administered at an injection volume of 5 µL for each. During the process, if the mouse demonstrated tail movement, the intrathecal injection was successful.
For intraperitoneal injection, MCC950 (1 mg/kg, 3 mg/kg, 10 mg/kg) or CY-09 (5 mg/kg, Sigma, SML2465) were injected by 1 mL sterile insulin syringe at 4 weeks after STZ injection.
To silence TET2 in DRG neurons, after three days of culture, cells were incubated with Opti-MEM™I (Gibco, 31985-070), Lipofectamine™ RNAiMAX (Invitrogen, 13778-150), and TET2-siRNA or Scr-siRNA for 12 h. Then the medium was changed to HG culturing medium for an additional 24 h.
RNA-sequence analysis
The total RNA was extracted from the DRG sample using the TRIzol reagent [
34]. Agilent 2100 Bioanalyzer was used for determining the concentration of the extracted RNA. NanoDrop was employed to detect the purity of the extracted RNA. Sequencing libraries were generated using NEBNext
® Ultra™ RNA Library Prep Kit for Illumina
® (NEB, USA) by following the manufacturer’s recommendations. Index codes were added to attribute the sequences to the corresponding samples. Subsequently, the prepared libraries were sequenced on the Illumina Hiseq 2000/2500 platform (IGENECODE, Beijing). The expression level of each transcript was calculated through FPKM (fragments per kilobase of the transcript sequence per million base pairs sequenced). Hierarchical clustering was performed to analyze the differentially expressed genes (DEGs). When analyzing the RNA-seq data, we set the fold change above 1.5 with a probability above 0.75 and considered the level of mRNA changes significant. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, when the
p value was lower than 0.05, the signaling pathway was considered significantly enriched.
Statistical analyses
The data were expressed as mean ± SD and analyzed in GraphPad Prism software. Two-tailed Student’s t test followed by Tukey's multiple comparisons test was used to compare two groups. In order to compare multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or two-way ANOVA followed by Bonferroni’s post hoc test was performed. A p-value of < 0.05 was considered statistically significant. All experiments were repeated at least three independent times except the RNA-seq experiment.
Discussion
DNP poses a great challenge in clinical treatment due to its complex and incomprehensible underlying mechanism. Research on the modulating function of environmental factors on the pain system has recently evolved, and mounting evidence suggests epigenetic modifications to be one of these modulators [
35,
36]. In recent years, increasing studies have shown that aberrant DNA methylation is associated with the pain mechanism in the central nervous system [
37]. Here, we showed that methylation of the whole genomic DNA was downregulated and the expression of DNA demethylase TET2 was increased in peripheral DRG of STZ-diabetic mice. The upregulation of TET2 led to the increase of mRNA level of
Txnip and subsequent activation of the NLRP3 pathway, which contributed to the mechanical allodynia of STZ-diabetic mice.
DNA demethylation is usually mediated by ten-eleven translocation (TET) proteins, including TET1, TET2, and TET3. TET1-3 has redundant functions and are all involved in pain, but might respond to different stimulates. The studies on spared nerve injury (SNI) and spinal nerve ligation (SNL) animal models found that the levels of
TET3 mRNA in the DRG were significantly increased, while the levels of
TET1 and
TET2 exhibited no change [
38,
39], indicating that TET3 having a vital role in neuropathic pain. In another study, the overexpression of TET1 in the DRG significantly alleviates SNL-induced pain hypersensitivity [
40]. Moreover, the use of high levels of glucose to cultivate human mesangial cells (HMCs) or db/db diabetic mice was observed to increase the mRNA and protein levels of
TET2, while the levels of
TET1 and
TET3 exhibited no significant difference [
18]. These results taken together with our present findings indicate that under different pain conditions, the TET1-3 plays different roles in neuropathic pain, and TET2 has a higher sensitivity to stimulation with high glucose levels compared to TET1 and TET3. In this perspective, the involvement of TET2 in DNP appears plausible.
The function of TET2 is to catalyze the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally, 5-carboxycytosine (5caC). As revealed by immunofluorescence staining in the present study, TET2 was located mainly in the nucleus of the DRG neurons, especially in the NF200
+ neurons. Therefore, the nuclear localization of TET2 in neurons was consistent with its function. In addition to the nuclei of DRG neurons, research reported that TET2 was also located in the nuclei of non-neuronal cells [
38]. Although the authors did not perform immunostaining, they speculated that TET2 most probably colocalized with satellite and Schwann cells [
38]. In our current study, although we did not investigate the localization and changes of TET2 in satellites in DRG, we cultured mouse primary DRG neurons and confirmed our in vivo results in vitro, indicating that TET2 regulates
Txnip expression mainly in DRG neurons. Agreeing with our results, another study reported that in DRG, NLRP3 was primarily located in the neuron nuclei and not in the satellite glial cells [
9]. NLRP3 is the key molecule within the inflammasome. Abundant evidence demonstrates that NLRP3 inflammasome is involved in pain conditions [
41]. Upon activation, NLRP3 inflammasome produces cleaved-IL-1β from pro-IL-1β, and cleaved-IL-1β is an inflammatory cytokine capable of inducing pain [
41‐
43]. Although the involvement of NLRP3 inflammasome in DNP has been reported in several studies [
11,
44], the epigenetic mechanisms of upregulation/activation of NLRP3 inflammasome in DNP remain unclear so far.
Studies reported high levels of glucose to upregulate the expressions of TET2 and NLRP3 [
18,
19]. In the GM-CSF-mediated monocyte-to-macrophage differentiation, certain inflammasome genes, such as
IL-1β,
NLRC5, and
AIM2, undergo DNA demethylation, and
TET2-siRNA is capable of impairing this demethylation and thereby impede the expression of the inflammasome-associated genes [
20]. Another recent research also reveals that in cerebral ischemia/reperfusion, TET2 demethylated LncRNA TUG1, then subsequently promoted NLRP3 inflammasome and contributed to the inflammatory response [
45]. These findings suggest that TET2 might be involved in the upregulation of NLRP3 inflammasome expression/activation. However, there were also research that suggested that TET2 can inhibit the expression of NLRP3 [
46,
47]. Therefore, the regulation of TET2 on NLRP3 inflammasome is still uncertain. In the present study, we observed that the protein expressions of both TET2 and NLRP3 were upregulated in the STZ-diabetic mice DRG, however, the mRNA level of
NLRP3 was neither upregulated in high glucose-incubated DRG neurons in vitro, nor changed in the
TET2-siRNA-treated DRGs in vitro and in vivo. TXNIP is an endogenous activator of the NLRP3 inflammasome, and studies had indicated that TXNIP/NLRP3 pathway contributed to the pain [
48,
49]. In the present study, we found that
TET2-siRNA could downregulate the gene expression of
Txnip and relieve DNP. These results indicated that TET2 was involved in the upregulation of TXNIP, which then activated the NLRP3 inflammasome to contribute to DNP
.
Studies found that the expression of NLRP3 in the sciatic nerves of DNP was increased [
10,
50] and high glucose-induced the expression of NLRP3 increase in Schwann cell line RSC96 [
50,
51]. These results suggested that NLRP3 in Schwann cells might also play a role in DNP. We also noticed that a report indicates that the upregulation of DNMT1 and DNMT3a mediated the increase of TXNIP in high glucose-stimulated RSC96 cells [
50]. We immunostained the expression of TET2 and NLRP3 on the sciatic nerve and Schwann cells, and found that although TET2 and NLRP3 colocalized with S100 (a Schwann cell marker), there was no markedly increased expression of TET2 and NLRP3 were noticed at four weeks post-STZ injection (data not shown). Thus, whether TET2–TXNIP–NLRP3 also plays a role in the sciatic nerve and Schwann cells still needs further investigation.
Besides DNA methylation, another possible basis of the epigenetic regulation of NLRP3 expression is the non-coding RNAs [
52], which may suppress or increase the gene expression by binding to the mRNA of the inflammasome. In 2012, Bauernfeind et al
. became the first to identify miR-233–3p as the human miRNA that could regulate NLRP3 [
53]. Since then, an increasing number of miRNAs are being confirmed to have a vital role in regulating the expression of NLRP3, such as miR-17–5p, miR-7, and miR-133a [
54‐
56]. In the present study, RNA-sequencing revealed that the expression of a few non-coding RNAs was also altered in STZ-diabetic mice DRG, such as snhg7, snhg20, lpw, etc. These non-coding RNAs could also be involved in the regulation of NLRP3, although further studies are required to confirm that.
Type 2 diabetes (non-insulin dependent) is more prevalent when compared to type 1 diabetes (insulin dependent). Although the expression of TET2 in DRG in type2 diabetes had not been investigated, studies revealed that the expression of NLRP3 in DRG in type 2 diabetes was increased [
11]. High glucose is a common and crucial factor in type 2 or type 1 diabetes pathologies. In our research, although we only used type 1 diabetes mouse model, we believe that the DNP in type 2 diabetes mouse would share the same TET2–TXNIP–NLRP3 axis mechanisms. We also found that gender does not affect the expression of TET2, TXNIP, and NLRP3. These results further indicated that TET2 upregulation NLRP3 through
Txnip depended more on high glucose, but not other factors.
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