Rumex japonicus Houtt. Protects Dopaminergic Neurons by Regulating Mitochondrial Function and Gut-Brain Axis in In Vitro and In Vivo Models of Parkinson's Disease

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Rumex japonicus Houtt. (RJ) has been used to treat gastrointestinal and inflammatory diseases in East Asia. However, it is unknown whether RJ can prevent PD. We investigated the neuroprotective effects of RJ in cellular and animal PD models, focused on mitochondrial function and the gut-brain axis. SH-SY5Y cells were treated with RJ (0.01 mg/mL) for 24 h, after which they were treated with the 1-methyl-4-phenylpyridinium ion (MPP⁺). MPP⁺-induced apoptosis increased mitochondrial reactive oxygen species and decreased ATP, PINK1, and DJ-1, which were inhibited by RJ. Ten-week-old C57BL/6N male mice were treated with 30 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 5 days and orally administered 50 or 100 mg/kg of RJ for 14 days. RJ alleviated MPTP-induced behavioral impairment, dopaminergic neuronal death, and mitochondrial dysfunction in the substantia nigra (SN) and suppressed the MPTP-induced increase in lipopolysaccharide, interleukin-1β, tumor necrosis factor-α, α-synuclein, and apoptotic factors in the SN and colon. Moreover, RJ inhibited the MPTP-mediated disruption of the tight junction barrier in the colon and blood-brain barrier of mice. Therefore, RJ alleviates MPTP-induced inflammation and dopaminergic neuronal death by maintaining mitochondrial function and tight junctions in the brain and colon.

Keywords: MPP+; MPTP; Parkinson’s disease; Rumex japonicus Houtt.; SH-SY5Y; gut–brain axis; neurodegeneration; neuroinflammation; tight junction; α-synuclein.

Figure 1

Cell viability and mitochondrial generation of ATP and reactive oxidative species (ROS) in Rumex japonicus Houtt. (RJ)-treated SH-SY5Y cells. (A,B) Cell viability of SH-SY5Y cells exposed to various doses of RJ for 24 h and 1 mM 1-methyl-4-phenylpyridinium ion (MPP⁺) for 24 h together with the RJ. (C) The levels of ATP. (D,E) The levels of mitochondrial ROS in MPP⁺-treated cells. The experiment was repeated 3 times and 5 figures per group were quantified and are presented as the mean ± standard deviation. Values are presented as the mean ± standard deviation. Mean values were significantly different (** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the group treated by MPP⁺ alone; ⁺⁺ p < 0.01 and ⁺⁺⁺ p < 0.001, compared with the group treated by RJ alone) as determined by Tukey’s honestly significant difference test. Scale bar = 50 μm.

Figure 2

Rumex japonicus Houtt. (RJ) attenuated 1-methyl-4-phenylpyridinium ion (MPP⁺)-mediated reduction in Parkinson’s disease (PD)-related mitochondrial proteins in SH-SY5Y cells. (A–D) Cells treated with RJ with/without MPP⁺ were stained with antibodies of PINK1, Parkin, and DJ-1. The immunofluorescence experiment was repeated 3 times and 5 figures per group were quantified. (E–H) Changes in PINK1, Parkin and DJ-1 were confirmed by Western blotting (n = 3). Values are presented as the mean ± standard deviation. Mean values were significantly different (* p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the group treated by MPP⁺ alone; ⁺⁺ p < 0.01 and ⁺⁺⁺ p < 0.001, compared with the group treated by RJ alone) as determined by Tukey’s honestly significant difference test. Scale bar = 50 μm.

Figure 3

Rumex japonicus Houtt. (RJ) inhibited 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced apoptosis in SH-SY5Y cells. (A–D) Apoptosis of the cells treated with RJ with/without MPP⁺ were confirmed by cytometry and quantified in the early, late, and total stages. (E–I) The expressions of Bcl-2, Bax, cytochrome c, and caspase-3 were confirmed by Western blotting. Values are expressed as the mean ± standard deviation (n = 3). Mean values were significantly different (* p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the group treated by MPP⁺ alone; ⁺ p < 0.05, ⁺⁺ p < 0.05 and ⁺⁺⁺ p < 0.001, compared with the group treated by RJ alone) as determined by Tukey’s honestly significant difference test.

Figure 4

Rumex japonicus Houtt. (RJ) alleviated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor dysfunction in mice with Parkinson’s disease. (A) In vivo study design of Parkinson’s disease. The behavior changes were confirmed by the pole test (B–E) and the rotarod test (F–I). Comparison between the groups on day 13 in the pole test (J) and the rotarod test (K). Values are expressed as the mean ± standard error of mean (n = 6). Mean values were significantly different as determined by the paired t-test (* p < 0.05 and ** p < 0.01, between day 0 and 5; ^# p < 0.05, ^## p < 0.01, between day 5 and 13) or Duncan’s multiple range tests (different lowercase letters (a, b) were significantly different (p < 0.05)).

Figure 5

Rumex japonicus Houtt. (RJ) attenuated the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and Parkinson’s disease (PD)-related mitochondrial proteins in the SN of PD mice. (A,B) Changes in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of tyrosine hydroxylase (TH)-positive cells in immunohistochemical observation. (C) The protein expressions of TH in the SN. (D–G) The mitochondrial factors in SN including PINK1, Parkin and DJ-1. Values are expressed as the mean ± standard deviation (n = 3). Mean values were significantly different (*** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the MPTP group; ⁺ p < 0.05, ⁺⁺ p < 0.01 and ⁺⁺⁺ p < 0.001, compared with the RJ-L group) by Tukey’s honestly significant difference test. Scale bar = 200 μm.

Figure 6

Rumex japonicus Houtt. (RJ) inhibited 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in the substantia nigra (SN) and the colon of mice. (A–E) The protein expressions of apoptotic factors in the SN, including Bcl-2, Bax, cytochrome c, and cleaved caspase-3. (F–J) The same apoptotic factors of the SN and colon. Values are expressed as the mean ± standard deviation (n = 3). Mean values were significantly different (* p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the MPTP group; ⁺ p < 0.05 and ⁺⁺ p < 0.01, compared with the RJ-L group) as determined by Tukey’s honestly significant difference test.

Figure 7

Rumex japonicus Houtt. (RJ) suppressed collapse of tight junction (TJ) barrier in the colon of a mouse model of Parkinson’s disease. (A–C) Changes in colonic TJs including ZO-1 and occludin in immunofluorescence assays. (D–F) The protein expressions of ZO-1 and occludin. Values are expressed as mean ± standard deviation (n = 3). Mean values were significantly different (* p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the normal group; ^## p < 0.01 and ^### p < 0.001, compared with the MPTP group; ⁺⁺⁺ p < 0.001, compared with the RJ-L group) as determined by Tukey’s honestly significant difference test. Scale bar = 50 μm.

Figure 8

Rumex japonicus Houtt. (RJ) decreased the levels of lipopolysaccharide (LPS) and pro-inflammatory cytokines in the colon, serum, or substantia nigra (SN) of a mouse model of Parkinson’s disease. (A–C) The changes in the level of LPS in the colon, serum, and SN. (D,E) Interleukin (IL)-1β in the colon and the SN. (F,G) Tumor necrosis factor (TNF)-α in the colon and the SN. Values are expressed as mean ± standard deviation (n = 3). Mean values were significantly different (* p < 0.05 and ** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, compared with the MPTP group) as determined by Tukey’s honestly significant difference test.

Figure 9

Rumex japonicus Houtt. (RJ) attenuated the increase in α-synuclein levels in the colon, serum, and substantia nigra (SN) of a mouse model of Parkinson’s disease. (A,B) The changes in the level of colonic α-synuclein by immunofluorescence assay. (C–E) The levels of α-synuclein in the colon, serum, and SN by enzyme-linked immunosorbent assay. Values are expressed as mean ± standard deviation (n = 3). Mean values were significantly different (** p < 0.01 and *** p < 0.001, compared with the normal group; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the MPTP group) as determined by Tukey’s honestly significant difference test. Scale bar = 100 μm.

Figure 10

Rumex japonicus Houtt. (RJ) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated weakness of the blood–brain barrier (BBB) in a mouse model of Parkinson’s disease (PD). (A–D) The protein expressions of the BBB-related proteins, including VE-cadherin, occludin, and claudin-5 in the SN of PD mice. Values are expressed as mean ± standard deviation (n = 3). Mean values were significantly different (** p < 0.01 and *** p < 0.001, compared with the normal group; ^### p < 0.001, compared with the MPTP group; ⁺ p < 0.05 and ⁺⁺⁺ p < 0.001, compared with the RJ-L group) as determined by Tukey’s honestly significant difference test.