Liquiritin ameliorates painful diabetic neuropathy in SD rats by inhibiting NLRP3-MMP-9-mediated reversal of aquaporin-4 polarity in the glymphatic system

Abstract

Background: Despite advancements in diabetes treatment, the management of Painful Diabetic Neuropathy (PDN) remains challenging. Our previous research indicated a significant correlation between the expression and distribution of Aquaporin-4 (AQP4) in the spinal glymphatic system and PDN. However, the potential role and mechanism of liquiritin in PDN treatment remain uncertain.

Methods: This study established a rat model of PDN using a combination of low-dose Streptozotocin (STZ) and a high-fat, high-sugar diet. Rats were treated with liquiritin and MCC950 (an NLRP3 inhibitor). We monitored fasting blood glucose, body weight, and mechanical allodynia periodically. The glymphatic system's clearance function was evaluated using Magnetic Resonance Imaging (MRI), and changes in proteins including NLRP3, MMP-9, and AQP4 were detected through immunofluorescence and Western blot techniques.

Results: The rats with painful diabetic neuropathy (PDN) demonstrated several physiological changes, including heightened mechanical allodynia, compromised clearance function within the spinal glymphatic system, altered distribution of AQP4, increased count of activated astrocytes, elevated expression levels of NLRP3 and MMP-9, and decreased expression of AQP4. However, following treatment with liquiritin and MCC950, these rats exhibited notable improvements.

Conclusion: Liquiritin may promote the restoration of AQP4 polarity by inhibiting NLRP3 and MMP-9, thereby enhancing the clearance functions of the spinal cord glymphatic system in PDN rats, alleviating the progression of PDN.

Keywords: aquaporin-4; glymphatic system; liquiritin; matrix metalloproteinase-9; painful diabetic neuropathy.

FIGURE 1

Experimental flow chart. The group LQ was administered liquiritin orally each day, while the group MCC950 received daily intraperitoneal injections of MCC950. The group C and group PDN rats were respectively administered gastric gavage and intraperitoneal injections of saline solution. A month after treatment, 4 rats from each group underwent MRI examinations. One week after the examination, spinal cords were extracted for Western Blot (WB) analysis, and the remaining 6 rats underwent immunofluorescence detection.

FIGURE 2

The trend of body weight, blood glucose and PWT change in rats. (A) The trend of body weight in Group C, Group PDN, Group LQ and Group MCC950. (B) The trend of blood glucose in Group C, Group PDN, Group LQ and Group MCC950. (C) The trend of PWT in Group C, Group PDN, Group LQ and Group MCC950. The one-way analysis of variance (ANOVA) was used to compare means between groups. The repeated-measures ANOVA was used to compare the PWT. Post hoc analyses were conducted using the Bonferroni correction method. Refer to Figure 1 for the meaning of the grouping abbreviation. Values were presented as mean ± SEM. Group C compared with group PDN, ^* p < 0.05, ^** p < 0.01, ^**** p < 0.0001; Group C compared with group MCC950, ^# p < 0.05, ^### p < 0.001, ^#### p < 0.0001; Group C compared with group LQ, ^{^^^} p < 0.001, ^{^^^^} p < 0.0001.

FIGURE 3

Metabolic image of Gd-DTPA at spinal cord lumbar enlargement in rats (Initial–6 h). Initial: Before injection of Gd-DTPA; 15 min: Inject Gd-DTPA for 15 min; 30 min: Inject Gd-DTPA for 30 min; 1 h: Inject Gd-DTPA for 1 h; 1.5 h: Inject Gd-DTPA for 1.5 h; 2 h: Inject Gd-DTPA for 2 h; 2.5 h: Inject Gd-DTPA for 2.5 h; 3 h: Inject Gd-DTPA for 3 h; 6 h: Inject Gd-DTPA for 6 h. Refer to Figure 1 for the meaning of the grouping abbreviation.

FIGURE 4

Immunofluorescence results of AQP4 and GFAP. (A) Immunofluorescent staining was performed on the lumbar enlargement segments of the spinal cord in the C, PDN, LQ, and MCC950 groups of rats. Activated astrocytes were marked with GFAP and identified by green fluorescence. AQP4 protein was identified with red fluorescence, and cell nuclei were counterstained with DAPI (blue). Enlarged sections detailed the polarity reversal of AQP4 protein. Scale bar = 50 µm. (B) Quantitative analysis of fluorescence in activated astrocytes across the groups. (C) Quantitative analysis of fluorescence polarization of AQP4. Refer to Figure 1 for the meaning of the grouping abbreviation. Values were presented as mean ± SEM. Compared to the group PDN, ^* p < 0.05, ^*** p < 0.001, ^**** p < 0.0001.

FIGURE 5

Immunofluorescence results of AQP4 and CD31. (A) Immunofluorescent staining was performed on the lumbar enlargement segments of the spinal cord in the C, PDN, LQ, and MCC950 groups of rats. Blood vessels were marked with CD31 and identified by green fluorescence. AQP4 protein was identified with red fluorescence, and cell nuclei were counterstained with DAPI (blue). Enlarged sections detailed the polarity reversal of AQP4 protein. Scale bar = 50 µm. (B) Quantitative analysis of fluorescence polarization of AQP4. Refer to Figure 1 for the meaning of the grouping abbreviation. Values were presented as mean ± SEM. Compared to the group PDN, ^* p < 0.05, ^**** p < 0.0001.

FIGURE 6

Western blot results. (A) Western blot analysis of NLRP3, MMP-9 and AQP4 protein expression in Group C, Group PDN, Group LQ and Group MCC950. (B) Relative expression of NLRP3 protein in each group. (C) Relative expression of MMP-9 protein in each group. (D) Relative expression of AQP4 protein in each group. Refer to Figure 1 for the meaning of the grouping abbreviation. Values were presented as mean ± SEM. Compared with values in Group PDN, ^∗ p < 0.05, ^** p < 0.01 and ^*** p < 0.001.