Implication of lncRNA MSTRG.81401 in Hippocampal Pyroptosis Induced by P2X7 Receptor in Type 2 Diabetic Rats with Neuropathic Pain Combined with Depression

Abstract

Major depressive disorder (MDD) is a common complication of diabetes and is often observed alongside diabetic neuropathic pain (DNP) as a comorbidity in diabetic patients. Long non-coding RNA (lncRNA) plays an important role in various pathophysiological processes. The P2X7 receptor is responsible for triggering inflammatory responses, such as pyroptosis, linked to pain and depression. The aim of this study was to investigate the effect of lncRNA MSTRG.81401 on hippocampal pyroptosis induced by the P2X7 receptor in diabetic rats with DNP combined with MDD (DNP + MDD). Our results showed that the expression of lncRNA MSTRG.81401 was significantly elevated in the hippocampus of DNP + MDD rats compared with the control group. Following the administration of shRNA targeting lncRNA MSTRG.81401, a notable elevation in mechanical and thermal pain thresholds was observed in rats with comorbid DNP and MDD. Additionally, significant improvements in depression-like behaviors were evident in the open-field test (OFT), sucrose preference test (SPT), and forced swim test (FST). In the DNP + MDD rats, elevated levels in hippocampal P2X7 receptor mRNA and protein were observed, along with increased co-expression of P2X7 and the astrocytic marker glial fibrillary acidic protein (GFAP). Meanwhile, in DNP + MDD rats, the heightened mRNA expression of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), pyroptosis-related protein Gasdermin D (GSDMD), caspase-1, IL-1β, IL-18, and TNF-α was detected, in addition to increased serum levels of IL-1β, IL-18 and TNF-α. After shRNA treatment with lncRNA MSTRG.81401, the above abnormal changes in indicators for pyroptosis and inflammation were improved. Therefore, our study demonstrates that shRNA of lncRNA MSTRG.81401 can alleviate the pain and depression-like behaviors in diabetic rats associated with the comorbidity of DNP and MDD by inhibiting the hippocampal P2X7 receptor-mediated pyroptosis pathway and pro-inflammatory responses. This suggests that the P2X7R/NLRP3/caspase-1 implicated pyroptosis and inflammatory scenario may serve as a potential target for the management of comorbid DNP and MDD in diabetes.

Keywords: P2X7 receptor; diabetic neuropathologic pain; hippocampus; lncRNA; major depressive disorder; pyroptosis.

Figure 1

Effects of MSTRG.81401 shRNA on mechanical withdrawal threshold (MWT) (A), thermal withdrawal latency (TWL) (B) of comorbid diabetic neuropathic pain (DNP) and major depressive disorder (MDD) rats. Values are mean ± SEM, n =  6; * p <  0.05, ** p <  0.01 and *** p <  0.001 vs. Ctrl group; ### p < 0.001 vs. DNP + MDD group.

Figure 2

Effects of MSTRG.81401 shRNA on sucrose preference test (SPT) (A), open-field test (OFT) (B) and forced swimming test (FST) (C) of comorbid DNP and MDD rats; Trajectory maps show the open-field movement of rats (D). Values are mean ± SEM, n  =  6; * p <  0.05, ** p <  0.01, *** p <  0.001 vs. Ctrl group; ### p <  0.001 vs. DNP + MDD group.

Figure 3

Measurement of MSTRG.81401 expression levels in hippocampus by qPCR (A) and in situ hybridization (B). Values are mean ± SEM, n = 6; *** p < 0.001 vs. Ctrl group; ### p < 0.001 vs. DNP + MDD group. Black arrows indicate the immunostaining cells.

Figure 4

Measurements of P2X7 mRNA and protein levels in hippocampus and co-expression of P2X7 with GFAP. The levels of P2X7 mRNA were measured by qRT-PCR (A). P2X7 protein in hippocampus of experimental rats was assessed by Western blotting (B) and the analyzed results are shown in (C). Co-expression of GFAP and P2X7 in hippocampus was detected by immunofluorescence double labeling (D); Blue staining (DAPI) marks the nucleus, green staining (GFAP) marks the astrocyte, red staining marks P2X7, and the yellow signal is a combination of green and red signals. The scale bar represents 50 μm. The relative fluorescence intensity levels of GFAP and P2X7R co-expression are presented in (E). Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; ## p < 0.01, ### p < 0.001 vs. DNP + MDD group. White arrows indicate the immunostaining cells.

Figure 4

Measurements of P2X7 mRNA and protein levels in hippocampus and co-expression of P2X7 with GFAP. The levels of P2X7 mRNA were measured by qRT-PCR (A). P2X7 protein in hippocampus of experimental rats was assessed by Western blotting (B) and the analyzed results are shown in (C). Co-expression of GFAP and P2X7 in hippocampus was detected by immunofluorescence double labeling (D); Blue staining (DAPI) marks the nucleus, green staining (GFAP) marks the astrocyte, red staining marks P2X7, and the yellow signal is a combination of green and red signals. The scale bar represents 50 μm. The relative fluorescence intensity levels of GFAP and P2X7R co-expression are presented in (E). Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; ## p < 0.01, ### p < 0.001 vs. DNP + MDD group. White arrows indicate the immunostaining cells.

Figure 5

Expression of inflammasome/pyroptosis pathway proteins (A–G) and inflammatory factors (H–M) in hippocampus was examined by Western blotting. The bar graphs show the statistical data of relative expression levels of targeted molecules after being normalized by β-actin. NLRP3, GSDMD and ASC (A–D); procaspase-1 and cleaved-caspase-1 (E–G); TNF-α (H,I), IL-1β (J,K) and IL-18 (L,M). Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. DNP + MDD group.

Figure 5

Expression of inflammasome/pyroptosis pathway proteins (A–G) and inflammatory factors (H–M) in hippocampus was examined by Western blotting. The bar graphs show the statistical data of relative expression levels of targeted molecules after being normalized by β-actin. NLRP3, GSDMD and ASC (A–D); procaspase-1 and cleaved-caspase-1 (E–G); TNF-α (H,I), IL-1β (J,K) and IL-18 (L,M). Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. DNP + MDD group.

Figure 6

The serum concentrations of TNF-α (A), IL-18 (B) and IL-1β (C) were assessed using ELISA. Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; ## p < 0.01, ### p < 0.001 vs. DNP + MDD group.

Figure 7

Timeline of the model establishment and sample collection.