Total saponins from Trillium tschonoskii Maxim promote neurological recovery in model rats with post-stroke cognitive impairment

Abstract

Total saponins from Trillium tschonoskii Maxim (TSTT), a bioactive component of local natural herbs in the Enshi area, China, have been demonstrated to have functions of restoring cognitive capacity and promoting axonal regeneration post-stroke, but the mechanism of this process remains unclear. The hippocampus is a critical tissue for controlling learning and memory capacity, and the sonic hedgehog (Shh) signaling pathway plays a major role in the patterning and synaptic plasticity of hippocampal neural circuits. Therefore, we aimed to investigate whether TSTT could restore learning and cognitive functions by modulating the Shh pathway in rats with post-stroke cognitive impairment (PSCI). The ischemia model was established by permanent middle cerebral artery occlusion (MCAO) in 100 Sprague-Dawley (SD) rats, and the model rats were administered using TSTT (100 mg/kg) or donepezil hydrochloride as the positive control (daily 0.45 mg/kg, DON) for 4 weeks after the operation. As assessed by the Morris water maze test, the cognitive function of PSCI rats was significantly improved upon TSTT treatment. Meanwhile, the cerebral infarct volume reduced with TSTT, as shown by HE and TTC staining, and the number of Nissl bodies and dendritic spine density were significantly increased, as shown by Nissl and Golgi staining. In addition, TSTT upregulated PSD-95, SYN, and GAP-43, and inhibited neuronal apoptosis, as evidenced by increased Bcl-2 levels along with decreased Bax and caspase-3 expression. TSTT could also significantly upregulate Shh, Ptch1, Smo, and Gli1 proteins, indicating the activation of the Shh signaling pathway. Therefore, TSTT can protect PSCI rats by inhibiting apoptosis and promoting neuronal synaptic remodeling. The Shh pathway is also involved.

Keywords: Shh signaling pathway; apoptosis; neuronal synaptic; post-stroke cognitive impairment; total saponins from Trillium tschonoskii Maxim.

FIGURE 1

Morris water maze test results and representative swimming trajectory of rats from each group: (A) time to find the platform, (B) swimming distance, (C) escape latency, (D) the number of crossing platforms, (E) target quadrant dwell time, (F) swimming trajectory map for positioning navigation experiments, and (G) swimming trajectory map for space exploration experiments. ^** p < 0.01 vs. Sham group; ^## p < 0.01 vs. MCAO group; n = 15.

FIGURE 2

TTC staining and statistical results of brain tissue in rats from each group. (A) TTC staining after intragastric administration for 7 days. (B) TTC staining after intragastric administration for 28 days. (C) Quantification of the ischemic volume after intragastric administration for 7 days. (D) Quantification of the ischemic volume after intragastric administration for 28 days. (E) Comparison of the ischemic volume after 7 days of intragastric administration. (F) Comparison of the ischemic volume after 28 days of intragastric administration. ^** p < 0.01 vs. Sham group; ^## p < 0.01 vs. MCAO group; n = 3.

FIGURE 3

HE and Nissl staining after brain tissue perfusion in each group:(A) Representative map of brain tissue perfusion in each group. (B) HE staining of whole brain tissue; Scale bar = 1,000 μm. (C) HE staining of the hippocampal CA1 region; scale bar = 100 μm. (D) Nissl staining of the hippocampal CA1 region a; scale bar = 100 μm. (E) Nissl staining in the cortex and ischemic penumbra; scale bar = 50 μm.

FIGURE 4

TUNEL staining and Bcl-2, Bax, cl-caspase-3, and caspase-3 proteins in the hippocampal CA1 region of rats from each group: (A) TUNEL staining of brain tissue: scale bar = 1,000 μm; DAPI/TUNEL/Merge: scale bar = 50 μm. (B) Neuronal apoptosis rate in the hippocampal CA1 region of rats in each group. (C) Average optical density of the hippocampal CA1 region in each group. (D) Expression of Bcl-2, Bax, cl-caspase-3, and caspase-3 proteins. (E) Relative expression of Bcl-2. (F) Relative expression of Bax. (G) Relative expression of cl-caspase-3/caspase-3. ^** p < 0.01 vs. Sham group; ^## p < 0.01 vs. MCAO group; n = 3.

FIGURE 5

TSTT promoted synaptic remodeling in hippocampal neurons of PSCI rats. (A) PSD-95 immunofluorescence labeling in the hippocampal CA1 region; scale bar = 50 μm. (B) SYN immunofluorescence labeling in the hippocampal CA3 region; scale bar = 50 μm. (C) GAP-43 immunofluorescence labeling in the hippocampal CA3 region; scale bar = 50 μm. (D) Golgi staining of rats in each group; scale bar = 10 μm. (E) PSD-95, SYN, and GAP-43 protein expression in the hippocampus. (F) Population of PSD-95-positive cells. (G) Population of SYN-positive cells. (H) Population of GAP-43-positive cells. (I) Density of dendritic spines. (J) Relative expression of PSD-95. (K) Relative expression of SYN. (L) Relative expression of GAP-43. ^** p < 0.01 vs. Sham group; ^## p < 0.01 vs. MCAO group; n = 3.

FIGURE 6

TSTT promoted Shh/Gli1 co-localization in the hippocampal CA3 region and ischemic penumbra in PSCI rats. (A) Shh/Gli1 co-localization staining in the hippocampal CA3 region and ischemic penumbra, HP: scale bar = 500 μm; HP-Merge: scale bar = 50 μm; IP: scale bar = 200 μm; IP-Merge: 20 μm. (B) Expression of Shh, Ptch1, Smo, and Gli1 proteins in the hippocampus. (C) Population of Shh/Gli1-positive co-localization positive cells in the hippocampal CA3 area. (D) Population of Shh/Gli1-positive co-localization cells in the hippocampal IP area. (E) Relative expression of Shh. (F) Relative expression of Ptch1. (G) Relative expression of Smo. (H) Relative expression of Gli1. ^* p < 0.05 or ^** p < 0.01 vs. Sham group; ^# p < 0.05 or ^## p < 0.01 vs. MCAO group; n = 3.