Ginseng Gintonin Attenuates Lead-Induced Rat Cerebellar Impairments during Gestation and Lactation

Abstract

Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, improves brain functions and protects neurons from oxidative stress. However, little is known about the effects of gintonin against Pb-induced brain maldevelopment. We investigated the protective effects of gintonin on the developing cerebellum after prenatal and postnatal Pb exposure. Pregnant female rats were randomly divided into three groups: control, Pb (0.3% Pb acetate in drinking water), and Pb plus gintonin (100 mg/kg, p.o.). Blood Pb was increased in dams and pups; gintonin treatment significantly decreased blood Pb. On postnatal day 21, the number of degenerating Purkinje cells was remarkably increased while the number of calbindin-, GAD67-, NMDAR1-, LPAR1-immunoreactive intact Purkinje cells, and GABA transporter 1-immunoreactive pinceau structures were significantly reduced in Pb-exposed offspring. Following Pb exposure, gintonin ameliorated cerebellar degenerative effects, restored increased pro-apoptotic Bax, and decreased anti-apoptotic Bcl2. Gintonin treatment attenuated Pb-induced accumulation of oxidative stress (Nrf2 and Mn-SOD) and inflammation (IL-1β and TNFα,), restoring the decreased cerebellar BDNF and Sirt1. Gintonin ameliorated Pb-induced impairment of myelin basic protein-immunoreactive myelinated fibers of Purkinje cells. Gintonin attenuated Pb-induced locomotor dysfunctions. The present study revealed the ameliorating effects of gintonin against Pb, suggesting the potential use of gintonin as a preventive agent in Pb poisoning during pregnancy and lactation.

Keywords: cerebellar lead (Pb) poisoning; ginseng; gintonin; neuroprotection; pregnancy and lactation.

Figure 1

Experimental design and physiological changes after Pb alone or gintonin plus Pb. (A) The present study was performed as shown in the experimental design. (B) Body weights of dams (n = 3 per group) during gestation and offspring (n = 12 per group) on postnatal days (PND) 0, 7, 14, and 21 in the control, Pb, and Pb plus gintonin (Pb + GT) groups (body weight, p = 0.9676, one-way analysis of variance). Whole-brain and cerebellar weights of offspring on PND21 (wet brain weight, p < 0.0001, one-way analysis of variance; wet cerebellar weight, p = 0.0080, one-way analysis of variance). (C) Blood Pb levels in the dams and offspring PND21 (blood Pb level, p < 0.0001, one-way analysis of variance). * p < 0.05, compared to control group, # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate means ± standard error of mean.

Figure 2

Effects of gintonin on Pb-induced cerebellar Purkinje cell impairments. Nissl staining (A–C), immunohistochemistry for calbindin (CB) (D–F), and immunohistochemistry for Bax (G–I) in the cerebellum of offspring from control, Pb, and Pb plus gintonin (Pb + GT) groups. GCL, granular cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. Degenerating Purkinje cells (red arrow), CB-immunoreactive Purkinje cells, bax-immunoreactive cells (arrow) are detected in the Purkinje cell layer. (J) The numbers of degenerating Purkinje cells (p < 0.0001, one-way analysis of variance) and CB-immunoreactive Purkinje cells (p < 0.0001, one-way analysis of variance) in the cerebellum are expressed as percentages of the value in the control group (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate means ± standard error of the mean.

Figure 3

Effects of gintonin on Pb-induced cerebellar changes in various protein expression levels. Representative immunoblots for BDNF, Sirt1, MBP, GAD67, LPAR1, synaptophysin, NMDAR1, Bax, Bcl2, cleaved-caspase3, Nrf2, Mn-SOD, IL-1β, and TNFα (A,B) in the cerebellum of pups from control, Pb, and Pb plus gintonin (Pb + GT) groups at postnatal day 21 (PND21). Relative optical density (ROD) of each of immunoblot bands for BDNF (p = 0.0010, one-way analysis of variance), Sirt1 (p = 0.0020, one-way analysis of variance), MBP (p < 0.0001, one-way analysis of variance), GAD67 (p = 0.0022, one-way analysis of variance), LPAR1 (P = 0.0001, one-way analysis of variance), synaptophysin (p < 0.0001, one-way analysis of variance), NMDAR1 (p < 0.0001, one-way analysis of variance), Bax (p = 0.0018, one-way analysis of variance), Bcl2 (p < 0.0001, one-way analysis of variance), cleaved-caspase3 (p < 0.0001, one-way analysis of variance), Nrf2 (p < 0.0001, one-way analysis of variance), Mn-SOD (p < 0.0001, one-way analysis of variance), IL-1β (p < 0.0001, one-way analysis of variance), and TNFα (p < 0.0001, one-way analysis of variance) is demonstrated as a percentage of the value in the control group (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate the means ± standard error of the mean.

Figure 4

Effects of gintonin on Pb-induced changes in cerebellar LPAR1 and NMDA receptor expression. Immunohistochemistry of LPAR1 (A–C), NMDAR1 (D–F), and NMDAR2B (G–I) in the cerebellum of offspring from control, Pb, and Pb plus gintonin (Pb + GT) groups. LPAR1 and NMDAR1 are mainly detected in the Purkinje cell layer, and NMDAR2B is detected in the granular cell layer in the cerebellar cortex. The number of LPAR1-immunoreactive and NMDAR1-immunoreactive intact Purkinje cells is significantly reduced, while gintonin treatment ameliorated this reduction in the cerebellum. GCL, granular cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. (J) Numbers of LPAR1-immunoreactive Purkinje cells (p = 0.0031, one-way analysis of variance) and NMDAR1-immunoreactive Purkinje cells (p = 0.0042, one-way analysis of variance) are expressed as a percentage of the value in the control group in the cerebellar cortex (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate means ± standard error of the mean.

Figure 5

Effects of gintonin on Pb-induced changes in cerebellar GAD67 and GABA transporter1. Immunohistochemistry for GAD67 (A–C) and GABA transporter 1 (GABAT1) (D–F) in the cerebellum of offspring from control, lead (Pb), and Pb plus gintonin (Pb + GT) groups. Note that GAD67-immunoreactive Purkinje cells and GABAT1-positive pinceau structures (arrow) near Purkinje cells are detected in the cerebellar cortex and are significantly reduced in the Pb group; gintonin treatment attenuates these reductions in the Pb + GT group. GCL, granular cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. (G) The number of GAD67-positive Purkinje cells (p = 0.0017, one-way analysis of variance) and GABAT1-positive pinceau structures (p < 0.0001, one-way analysis of variance) is expressed as a percentage of the value in the control group in the cerebellar cortex (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate means ± standard error of mean.

Figure 6

Effects of gintonin on Pb-induced cerebellar MBP and Olig2. Immunohistochemistry of MBP (A–C) and Olig2 (D–F) in the cerebellum of offspring from control, Pb, and Pb plus gintonin (Pb + GT) groups. Note that MBP-positive myelinated fibers and Olig2-immunoreactive oligodendrocytes (arrow) are detected in the cerebellum. Pb-induced reduction in the number of Olig2-immunoreactive oligodendrocytes is significantly ameliorated by gintonin treatment. The intensity of MBP staining in white matter (WM) (B) decreases after Pb exposure, with gintonin treatment ameliorating these observed reductions. MBP, Myelin basic protein; GM-GCL, granular cell layer in gray matter; WM, white matter. Bar = 25 μm. (G) The number of Olig2-immunoreactive oligodendrocytes (p = 0.0011, one-way analysis of variance) in the cerebellar cortex is expressed as percentages of the value in the control group (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate the means ± standard error of the mean.

Figure 7

Effects of gintonin on Pb-induced locomotive impairments. Effect of Pb exposure and gintonin treatment on the bar holding test (A) and wire mesh ascending test (B) among offspring from control, Pb, and Pb plus gintonin (Pb + GT) groups. (A) The time during which the animal stayed on the bar (p < 0.0001, one-way analysis of variance) in the bar holding test. (B) The time spent to reach the top of the wire mesh (p = 0.0082, one-way analysis of variance) in the wire mesh ascending test (n = 12 per group). * p < 0.05, compared to control group; # p < 0.05, compared to Pb alone, Tukey’s post-hoc test. The bars indicate the means ± standard error of the mean.

Figure 8

Schematic illustration of gintonin-mediated anti-Pb effects. Cerebellar LPAR1 expression was reduced after Pb exposure, which was reversed by gintonin treatment. Gintonin-mediated anti-Pb effects could be mediated through three mechanisms: antioxidant stress, increased BDNF and Sirt1, and anti-apoptosis/inflammation via activation of LPAR1 signaling pathways. The convergence of these three beneficial mechanisms could increase the survival of cerebellar Purkinje cells and oligodendrocytes, facilitating synaptic transmission. These beneficial effects of gintonin contribute to the locomotive recovery in pups from Pb-poisoned dams.