Lamivudine improves cognitive decline in SAMP8 mice: Integrating in vivo pharmacological evaluation and network pharmacology

Abstract

The reverse transcriptase inhibitors such as lamivudine (3TC) play important roles in anti-ageing, but their effects on neurodegenerative diseases caused by ageing are not clear, especially on the functions of the nervous system such as cognition. In this study, we administered 3TC to senescence-accelerated mouse prone 8 (SAMP8) mice by gastric perfusion (100 mg/kg) for 4 weeks. Our results showed that 3TC significantly improved the ageing status of SAMP8 mice, especially the decline of cognitive ability evaluated by the Morris water maze test. To further investigate the molecular mechanisms of improving the ageing status of SAMP8 mice by 3TC, the qPCR and tissue staining methods were used to study the brain tissues (i.e., hippocampus and cortex) of mice, while the network pharmacology analysis was applied to investigate the potential targets of 3TC. The results showed that the mRNA levels of genes related to long interspersed element-1, type 1 interferon response, the senescence-associated secretion phenotype and the Alzheimer's disease in the hippocampus and cortex of SAMP8 mice were increased due to senescence, but this trend was reversed partially by 3TC. Results of histological studies showed that 3TC reduced the death of hippocampal neurons, while the results of network pharmacology analysis indicated that 3TC may exert its influence through multiple pathways, including the oestrogen signalling and the PI3K/Akt and neuroactive ligand-receptor interaction signalling pathways, which we have verified through in vitro experiments. These findings provide evidence for the therapeutic potential of 3TC in the treatment of neurodegenerative diseases.

Keywords: ageing; cognitive ability; lamivudine; long interspersed element-1; neurodegenerative diseases.

FIGURE 1

Experimental design and the effects of 3TC on improving relative weight loss due to ageing and senescence grading scores. (A) Flow chart of the pharmacological experiments to study the effect of 3TC on the cognitive ability of SAMP8 mice. (B) Schematic presentation of the animal experiment schedule. (C) The conceptual workflow for predicting the drug‐target interactions using Pharmmapper showing, from left to right, the molecular structure of 3TC, the 3D structural diagram of 3TC and the pharmacophore model predicted with 3TC binding to the oestrogen receptor with the highest normalized fit scores. (D) The growth pattern of body weight in each group of mice. (E) Changes in grading scores before and after the treatment of 3TC. SAMR1+Vehicle: 44‐week‐old SAMR1 mice vehicle‐treated for 4 weeks. SAMP8+Vehicle: 44‐week‐old SAMP8 mice vehicle‐treated for 4 weeks. SAMP8+3TC: 44‐week‐old SAMP8 mice 3TC‐treated for 4 weeks. Student's t‐test: *p < 0.05, **p < 0.01 and ***p < 0.001. ns: no statistical significance. Black asterisks represent comparison with the SAMR1+vehicle group. Red asterisks represent comparison with the SAMP8+vehicle group. The blue line marks the SAMP8+3TC group at the age of 44 and 48 weeks

FIGURE 2

The effects of 3TC treatment on improvements of the cognitive decline in SAMP8 mice. (A) Trajectory map of mice after entering the water from the second quadrant during the positioning navigation test on the 5th day. (B) Escape latency in the positioning navigation experiment for each group. (C) Swimming speed of mice in each group in behavioural tests. (D) Trajectory map of mice in the space exploration Experiment. (E) Percentage of total time spent in the target quadrant during the probe trials in each group. (F) The number of times that the mice crossed the platform in each group in the probe trials. (G) Escape time to target. Data are presented as mean ± SD. Student's t‐test: *p < 0.05, **p < 0.01 and ***p < 0.001. ns: no statistical significance. Black asterisks or "ns" represent comparison with the SAMR1+vehicle group. Red asterisks or "ns" represent comparison with the SAMP8+vehicle group. Each group consisted of a minimum of eight animals

FIGURE 3

Effects of 3TC treatment on the expressions of candidate genes in mouse brain tissues. Mice were treated with vehicle or 3TC continuously for 4 weeks. For all conditions, the expression of L1‐ORF1 and L1‐ORF2, three representative IFN‐I response genes (Ifna, Irf7 and Oas1), two Alzheimer disease‐related genes (PS‐1 and APP) and three representative SASP genes (Il6, Mmp3 and Pai1) in the hippocampus (A) and the cortex (B) were assessed by real‐time quantitative PCR with the expression profiling of genes shown in the histogram. Student's t‐test: *p < 0.05, **p < 0.01 and ***p < 0.001. ns: no statistical significance. Black asterisks or "ns" represent comparison with the SAMR1+vehicle group. Red asterisks or "ns" represent comparison with the SAMP8+vehicle group

FIGURE 4

3TC decreases neuronal injury in the hippocampus of SAMP8 mice. (A) Photomicrographs of H & E–stained hippocampus sections for each group of mice (bar = 50 μm). Black arrows mark concentrated nuclei, and blue arrows mark areas where neurons are severely lost and disarranged. (B) Identification of neuronal survival by Nissl staining in the hippocampus (bar = 20 μm). (C) The quantity of healthy neuronal cells in each visual field with densely stained Nissl bodies. (D) Images of apoptotic cells marked by TUNEL (Green) assay in each group of mice (bar = 20 μm). (E) Quantification of TUNEL‐positive cells in each visual field. Data are presented as mean ± SD. Student's t‐test: ***p < 0.001. Black asterisks represent comparison with the SAMR1+vehicle group. Red asterisks represent comparison with the SAMP8+vehicle group

FIGURE 5

Protein interplay diagram of the predicted targets of 3TC. (A) Protein interplay diagram of the 269 predicted targets of 3TC. The blue nodes indicate the 32 core target genes of 3TC. (B) Protein interplay diagram of the 32 core targets of 3TC. Proteins interaction is mimicked by String 9.1. The networks showing similar functions are labelled with the same colour at the nodes. The line thickness of the edges connecting the nodes indicates the strength of data support. The proteins labelled at the red nodes were significantly associated with fluid shear stress and atherosclerosis, the green nodes were mainly associated with the metabolism of xenobiotics by the cytochrome P450 pathway, and the blue nodes were involved in both calcium and cAMP signalling pathways

FIGURE 6

Gene Ontology (GO) enrichment and KEGG network analysis of target genes. (A) GO enrichment analysis of the 32 core target proteins of 3TC. The top 19 GO terms are presented in each of the three categories of GO terms (biological processes, cellular components and molecular functions). (B) The KEGG analysis of the 32 core targets of 3TC. The top 19 KEGG terms are presented. Bright red patches indicate high p‐values, and green patches indicate low p‐values. (C) Molecular models of 3TC binding to the predicted targets. Green labels indicate hydrogen bonds, small rods represent 3TC molecules, blue for amino acid residues, and orange for the ligand

FIGURE 7

Experiments in vitro confirmed the key molecules of 3TC against neuronal degeneration. Senescence of mouse neuron HT22 cells is induced by formaldehyde (FA). (A) Immunofluorescence detection of LINE‐1 ORF1p in control, FA and FA+3TC groups (bar = 20 μm). (B) Quantification of immunofluorescence analysis in (A). (C) Quantification of surviving neurons per ×400 field in various groups. (D) EGFR, p‐AKT1 and ADRB2 were detected by Western blot analysis. (E) Quantification of Western blot analysis in (D). Student's t‐test: ***p < 0.001. Black asterisks represent comparison with the control group. Red asterisks represent comparison with the FA group