[Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism].

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aß_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aß_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aß_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1ß(IL-1ß), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aß_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1ß, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aß_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1ß, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1ß, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aß and expression of p-Tau, thereby restoring the hippocampal morphological structure.

[Mechanism analysis of repeatedly steamed and sundried Rehmanniae Radix Praeparata in delaying brain aging in ovariectomized mice based on proteomics].

The present study explored the effect and mechanism of repeatedly steamed and sundried Rehmanniae Radix Praeparata(RRP) in delaying brain aging in ovariectomized mice. After ovariectomy, the mice were randomly divided into a model group, an estradiol valerate group(0.3 mg·kg~(-1)), and low-(1.0 g·kg~(-1)), medium-(2.0 g·kg~(-1)), and high-dose(4.0 g·kg~(-1)) RRP groups, and a sham operation group was also set up, with 15 mice in each group. One week after the operation, intragastric administration was carried out for 15 consecutive weeks. The step-down test and Morris water maze test were used to detect the behavioral changes of mice. HE staining and Nissl staining were used to observe the morphological changes of mouse brain tissues. Immunohistochemistry was used to detect the expression of Aß and ER_ß in mouse brain tissues. The serum estrogen levels and cholinesterase and cholinesterase transferase levels in brain tissues of mice were detected by assay kits. The extracted hippocampal protein was detected by the Nano-ESI-LC-MS system, identified by the Protein Discovery, and analyzed quantitatively and qualitatively by the SIEVE. The PANTHER Classification System was used for GO analysis and KEGG pathway enrichment analysis of the differential proteins. Compared with the sham operation group, the model group showed decreased learning and memory ability, shortened step-down latency(P<0.05), prolonged escape latency(P<0.05), reduced platform crossings and residence time in the target quadrant, scattered nerve cells in the hippocampus with enlarged intercellular space, increased expression of Aß-positive cells(P<0.05), declining expression of ER_ß-positive cells and estrogen level(P<0.05), and weakened cholinergic function(P<0.05). Compared with the model group, the RRP groups showed improved learning and memory ability, prolonged step-down latency(P<0.05), increased estrogen level(P<0.05), neatly arranged nerve cells in the hippocampus with complete morphology, declining Aß-positive cells, and elevated expression of ER_ß-positive cells. A total of 146 differential proteins were screened out by proteomics, and KEGG pathway enrichment yielded 75 signaling pathways. The number of proteins involved in the dopaminergic synapse signaling pathway was the largest, with 13 proteins involved. In summary, RRP can delay brain aging presumedly by increasing the level of estrogen, mediating the dopaminergic synapse signaling pathway, and improving cholinergic function.