Picrorhiza kurroa Prevents Memory Deficits by Inhibiting NLRP3 Inflammasome Activation and BACE1 Expression in 5xFAD Mice

Abstract

One of the most significant pathologies of Alzheimer's disease (AD), an irreversible and progressive neurodegenerative disease that causes cognitive impairment, is the neuroinflammation facilitating the accumulation of amyloid-β (Aβ) peptide. Hence, the inhibition of abnormal neuroinflammatory response is considered a promising therapeutic approach for AD. Picrorhiza kurroa Bentham, Scrophulariae (PK) is a medicinal herb that has been traditionally used for the treatment of various diseases, including inflammation. This study aims to report the significance of PK treatment in markedly improving spatial learning memory and dramatically decreasing Aβ levels in Tg6799 mice, also known 5xFAD mice, which have five familial AD (FAD) mutations. Remarkably, these effects correlated with reversal of disease-related microglial neuroinflammation, as evidenced by shifting microglia phenotypes from the inflammatory form to the anti-inflammatory form and inhibiting the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 inflammasome activity. Moreover, PK administration induced silent information regulator type1/peroxisome proliferator-activated receptor-γ signaling, resulting in a decrease of β-secretase 1 (BACE1) expression, which involved in Aβ production. Overall, this study suggests that PK exhibits a neuroprotective effect by inducing alternative activation of microglia and downregulating the BACE1 expression, thereby ameliorating the disease pathophysiology and reversing the cognitive decline related to Aβ deposition in AD mice.

Keywords: Alzheimer’s disease; NLRP3 inflammasome; Picrorhiza kurroa; amyloid-beta; inflammation; β-secretase 1.

Fig. 1

Picrorhiza kurroa Bentham, Scrophulariae (PK) ameliorates memory impairment in 5xFAD mice. (a) Protocol of the PK treatment in 5xFAD mice. (b) Escape latencies of wild-type (WT) or 5xFAD mice treated with saline or PK over 7 days (saline-treated WT mice, n = 15; PK-treated WT mice, n = 11; saline-treated 5xFAD mice, n = 15; PK-treated 5xFAD mice, n = 12). (c) Representative swimming paths on day 8 of training. (d–g) In probe trial day 8, spending time in the target quadrant (d), crossing the platform number (e), swim speed (f), and total distance (g) were recorded and analyzed. The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^#P < 0.05 versus saline-treated WT mice and *P < 0.05 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)

Fig. 2

Picrorhiza kurroa Bentham, Scrophulariae (PK) leads to reduce Aβ pathology in the hippocampus of 5xFAD mice. Brain sections from 7-month-old mice were stained with thioflavin S (ThS) and anti-6E10 antibody. (a) Representative images of ThS staining in the cortex and hippocampus; scale bar, 100 μm. (b) The quantification of ThS-stained area (n = 5–6 per group). (c) Immunofluorescence images of 6E10 staining in the cortex and hippocampus; scale bar, 100 μm. (d) The quantification of the 6E10-positive area (n = 5–6 per group). (e) Analysis of soluble and insoluble Aβ_1–42 levels from the mice hippocampus using ELISA kits (n = 6 per group). The statistical analysis included the Student’s t test. *P < 0.05 and **P < 0.01 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)

Fig. 3

Picrorhiza kurroa Bentham, Scrophulariae (PK) inhibits microglia activation, but not the astrocyte activation in the hippocampus of 5xFAD mice. Brain sections from 7-month-old mice were stained with anti-GFAP (anti-glial fibrillary acidic protein) and Iba-1 antibody. Immunofluorescence images of GFAP (a) and Iba-1 (e) staining in the hippocampus; scale bar, 100 μm. (b, f) The quantification of GFAP-positive- and Iba-1-positive cell area (n = 4–6 per group). Mouse brain lysates were analyzed for GFAP (c) and Iba-1 (g) levels using the western blot analysis. The quantification of GFAP (d) and Iba-1 (h) levels (n = 5–6 per group). The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^###P < 0.001 versus saline-treated wild-type (WT) mice and *P < 0.05 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)

Fig. 4

Picrorhiza kurroa Bentham, Scrophulariae (PK) modulates microglia phenotypes in the hippocampus of 5xFAD mice. The inflammatory (a–c) and anti-inflammatory (e–g) cytokines were measured in the hippocampus with quantitative qRT-PCR (n = 5–6 per group). (d) The protein level of IL-1β was analyzed in hippocampus lysate by the ELISA kit (n = 5–6 per group). The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^###P < 0.001 versus saline-treated wild-type (WT) mice and *P < 0.05 and **P < 0.01 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)

Fig. 5

Picrorhiza kurroa Bentham, Scrophulariae (PK) inhibits the NLRP3 inflammasome pathway in the hippocampus of 5xFAD mice. (a) The western blot analysis of NLRP3, ASC, and caspase-1 (Casp1) levels in the hippocampus of 7-month-old mice. (b–d) The quantification of NLRP3 (b), ASC (c), pro-Casp1, and cleaved Casp1 (d) were normalized to β-actin (n = 5–6 per group). The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^#P < 0.05 and ^##P < 0.01 versus saline-treated wild-type (WT) mice and *P < 0.05 and **P < 0.01 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)

Fig. 6

Picrorhiza kurroa Bentham, Scrophulariae (PK) do not affect Aβ-degrading enzymes expression in the hippocampus of 5xFAD mice. (a, b) The protein expression of insulin-degrading enzyme (IDE) (a) and neprilysin (b) were determined by the western blot analysis (n = 5–6 per group). (c, d) The mRNA expression of IDE (c) and neprilysin (d) were measured by qRT-PCR (n = 5–6 per group). The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^#P < 0.05 and ^###P < 0.001 versus saline-treated wild-type (WT) mice. Values are mean ± standard error of the mean (SEM)

Fig. 7

Picrorhiza kurroa Bentham, Scrophulariae (PK) inhibits amyloid precursor protein (APP) processing in the hippocampus of 5xFAD mice. (a) The western blot analysis of APP, C99, and presenillin1 levels in the hippocampus of 7-month-old mice. (b–d) The quantification of APP (d), C99 (c), and presenillin1 (d) were normalized to β-actin (n = 5–6 per group). (e) The mRNA of BACE1 was measured by qRT-PCR. (f) The western blot analysis of Pro-BACE1 and BACE1 levels in the hippocampus of 7-month-old mice. (g, h) The quantification of Pro-BACE1 (g) and BACE1 (h) were normalized to β-actin (n = 5–6 per group). The statistical analysis included the one-way analysis of variance and Tukey’s post hoc test. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus saline-treated wild-type (WT) mice and *P < 0.05 and **P < 0.01 versus saline-treated 5xFAD mice. Values are mean ± standard error of the mean (SEM)