Lamivudine Improves Cognitive Decline in SAMP8 Mice: Integrating in Vivo Pharmacological Evaluation And Network Pharmacology Ⅱ
May 29, 2023
3 | RESULTS
3.1 | 3TC reverses body weight loss caused by aging and improves the senescence score
The body weight changes of the mice in different experimental groups are shown in Figure 1D. The results showed that all mice gained body weight in 4 weeks. Under a standard diet, SAMP8 mice treated with 3TC gained weight faster than those treated with vehicles. The results of the senescence scores before and after the treatments of 3TC or vehicle showed that the average senescence scores of SAMP8 mice were relatively higher, while the reversed trend was achieved by 3TC treatment (Figure 1E). The results of the senescence-related scores of each group of mice collected on the last day of the experiment showed that SAMP8 mice treated with 3TC showed significant improvements in several senescence scores, including the glossiness of skin (p < 0.01, t = 3.901), the corneal opacity (p < 0.01, t = 4.753) and the ulcer of the cornea (p < 0.05, t = 6.125) (Supplementary Table S1).
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3.2 | 3TC improves the cognitive decline of SAMP8 mice
The Morris water maze tests were carried out to evaluate the cognitive effects of 3TC (Figure 2). Results of the trajectories and the directional navigation experiments for five consecutive days demonstrated that SAMR1 mice showed significantly lower escape latencies than those of SAMP8 mice (p < 0.05, t = 3.194) (Figure 2A–B). However, on the third and the fifth days, the escape latencies of the 3TC-treated SAMP8 mice were significantly shorter in comparison with those vehicle-treated SAMP8 mice (p < 0.05, t = 3.115), while no statistical significance was revealed in comparison with the SAMR1 group. Although the swimming speed of SAMP8 mice treated with 3TC on the first day was significantly faster than that of the other two groups, no significant difference was detected among the three experimental groups in the next 4 days of the experiments (Figure 2C).
The results of the probe trials and the trajectory on the 6th day showed that both the SAMR1 and the 3TC-treated SAMP8 group spent more time in the target quadrant than the vehicle-treated SAMP8 group (p < 0.05, t = 4.512; Figure 2D–E). Moreover, the numbers of crossings were also greater in both the SAMR1 mice and the 3TC-treated SAMP8 group than that in the vehicle-treated SAMP8 group, with a statistically significant difference revealed between the groups with and without the treatment of 3TC (p < 0.05, t = 5.79; Figure 2F). The vehicle-treated SAMP8 group showed the highest latencies to find the platform under the water among all groups with a statistically significant difference revealed between the groups treated and not treated with 3TC (p < 0.05, t = 4.415; Figure 2G).
FIGURE 2 The effects of 3TC treatment on improvements of 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 behavioral 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 a comparison with the SAMR1+vehicle group. Red asterisks or "ns" represent a comparison with the SAMP8+vehicle group. Each group consisted of a minimum of eight animals
3.3 | Target genes of 3TC revealed by the real-time quantitative PCR
Previous studies have shown that 3TC can reduce the SASP and IFN-I response signals to the smooth muscle cells of aging mice, but there is no report on the associated genes of neurological degenerative disease in brain tissue. To explore the effect of 3TC on the mice's brain, we investigated the expression of ten candidate genes of interest in the hippocampus and cortex of mice using real-time quantitative PCR. In the hippocampus (Figure 3A), the significantly up-regulated genes for aging (groups of SAMR1 vs. SAMP8 treated with vehicle) included one transposon-related gene (L1-ORF1, p < 0.05), three IFN-I response genes (Ifna, Irf7 and Oas1, p < 0.05), two AD-related genes (PS-1 and APP, p < 0.01) and three representative SASP genes (Il6, Mmp3 and Pai1, p < 0.01), while the significantly down-regulated genes for 3TC treatment (groups of SAMP8 treated with vehicle vs. SAMP8 treated with 3TC) included one transposonrelated gene (L1-ORF2, p < 0.05), two IFN-I response genes (Ifna and Oas1, p < 0.001) and three representative SASP genes (Il6, Mmp3 and Pai1, p < 0.01). Similar results were obtained in the cortex, though the up-regulation of the gene related to AD was decreased by the treatment of 3TC in the cortex in comparison with the hippocampus (p < 0.05; Figure 3B).
3.4 | 3TC attenuates morphological abnormalities and loss of neurons in the hippocampus of SAMP8 mice induced by aging
We applied both H & E and Nissl staining methods to investigate the morphological changes caused by 3TC in the hippocampus, which was the key area of cognition and memory. In comparison with the group of SAMR1 treated with a vehicle, the group of SAMP8 treated with a vehicle showed an increased clearance rate of hippocampal neurons, slightly disordered arrangement of cells, nuclear concentration found in some neurons and mild hippocampal edema. These damages were improved in the 3TC-treated groups (Figure 4A). The losses of Nissl-positive viable neuronal cells with typical neuropathological change were observed in the SAMP8 group based on Nissl staining, while these changes were partially reversed in the group of SAMP8 treated with 3TC (Figure 4B–C). The neuron death in the hippocampus was also detected by TUNEL staining with the most and the least amounts of TUNEL-positive cells (green) observed in the group of SAMP8 treated with vehicle and the SAMR1 group treated with vehicle, respectively, while the group of SAMP8 treated with 3TC was revealed significantly less than the group of SAMP8 treated with vehicle (Figure 4D–E).
3.5 | Putative targets of 3TC
The network pharmacology analysis revealed a total of 269 proteins as the predicted targets for 3TC with 164 identified by the Pharmmapper database and 105 by the ChEMBL database, respectively (Supplementary Table S3). The PPI network was constructed based on the target genes of 3TC using the String database with a total of 32 core nodes in the network identified as the key targets of 3TC in the SAMP8 mice (Figure 5A). We used String 9.1 to mimic the interactions between key target proteins of 3TC to identify three groups of proteins with similar functions (Figure 5B).
3.6 | Enrichment Analysis of the 3TC Key Target Networks
3.6.1 | GO Analysis
The GO enrichment analysis of the target genes identified under the treatment of 3TC using STRING revealed a total of 550 biological processes with p < 0.05 (Figure 6A; Supplementary Table S4), such as the response to acid chemical, the response to reactive oxygen species and the negative regulation of the apoptotic process. The GO analysis also identified a total of 23 cellular components with p < 0.01, such as the endomembrane system, cytoplasmic vesicle, vesicle, cytoplasmic part, and the plasma membrane part (Supplementary Table S5), while a total of 69 molecular functions identified by GO analysis were grouped in a total of 19 categories (Supplementary Table S6). It was worth noting that the tau protein binding term, which was thought to be linked to Alzheimer's disease, showed a significant relationship with 3TC (p < 0.05).
3.6.2 | Metabolic pathways of the 3TC target networks
A total of 109 significant treatment pathways were screened by KEGG analysis (p < 0.05; Figure 6B; Supplementary Table S7) with many pathways associated with cancers, including the pathways in cancer, chemical carcinogenesis, prostate cancer, proteoglycans in cancer and the microRNAs in cancer. Three pathways revealed to be related to neuroinflammation, cell death and neuronal signal transduction included estrogen signaling (p < 0.0001), the phosphorinositide 3-kinase and protein kinase B (PI3K/Akt) (p < 0.0001), and the neuroactive ligand-receptor interaction signaling (p < 0.001) pathways.27–29
3.6.3 | Binding mode
Docking studies were performed between 3TC and three selected potential targets, that is EGFR representing the estrogen signaling pathway, AKT1 representing the P13K/Akt signaling pathway and ADRB2 representing the neuroactive ligand-receptor interaction signaling pathway. The results of binding mode analysis showed (Figure 6C) that the amino acid residues Thr854, Asn842, Arg841 and Glu762 of the receptor protein EGFR interacted with small molecules of 3TC ligands to form hydrogen bonds, while amino acid residues Lys55, Val40, Ala53, Met111, Val158, Asn114, Leu168, Ser155, Asp169, Lys153, Asn156s and Gly35 formed hydrophobic interactions with small molecules of the 3TC ligand. Amino acid residues Asp292, Glu191, His194, Glu198s and Thr195 of the receptor protein AKT1 formed hydrogen bond interaction with 3TC, while amino acid residues Phe179, Leu295, Glu198 and Lys179 formed hydrophobic interaction with 3TC. The amino acid residues Asp331 and Phe332 of ADRB2 interacted with 3TC to form a hydrogen bond, while the amino acid residues Phe336, Ala335, Val54, Ala57, Ile58, Leu64, Asn69, Ile72 and Pro330 of ABRB2 formed hydrophobic interaction with 3TC. These results of docking studies provided evidence of 3TC binding to its targets, which was important to understand the mechanism of drug action. We further conducted in vitro experiments to verify the results of the bioinformatics analysis
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 a comparison with the SAMR1+vehicle group. Red asterisks or "ns" represent a comparison with the SAMP8+vehicle group.
3.7 | 3TC protects HT22 cells from FA-induced cytotoxicity by regulating a variety of molecules
To explore the effects of 3TC as a reverse transcriptase inhibitor on senescent neurons in vitro, we induced the senescence in HT22 cells with FA. The results showed that LINE-1 was activated in the senescent neurons, and this trend was reversed by 3TC (Figure 7A–B). In addition, 3TC showed an antagonistic effect on cell death induced by FA (Figure 7C). As expected, stimulation of TH22 cells with FA for 24 h resulted in a significant inhibition in the expression of EGFR, p-AKT1 and ADRB2 (p < 0.001), compared with the untreated control, while co-treatment with 3TC led to a significantly increased expression of FAinduced EGFR, p-AKT1 and ADRB2 (p < 0.001; Figure 7D,E).
4 | DISCUSSION
Cognitive declines, especially memory loss, are usually associated with natural aging. The effects of aging are particularly pronounced in the prefrontal cortex and hippocampus, the brain areas that are critically involved in cognition and mood.30 The SAMP8 mice were derived from the SAM-P/2 line as a rapidly aging mouse model of dementia. A previous study showed that the SAMP8 mice exhibited age-related cognitive decline with a short lifespan, showing progressive learning and memory deficits as well as neuropathological hallmarks of dementia in the prefrontal cortex and hippocampus.31 The lifespan of SAMP8 mice is almost half of that of SAMR1, which were used as the controls for the SAMP8.32 In our study, the SAMP8 mice were selected as an ideal animal model to investigate the decline of cognitive function during aging, with the homologous normally developing mice SAMR1 used as an anti-senescence control 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 a comparison with the SAMR1+vehicle group. Red asterisks represent a comparison with the SAMP8+vehicle group.
Aging usually causes the body to lose weight due to bone wasting, muscle atrophy and dysfunction in the digestive system.33 Our results showed that 3TC partially alleviated the trend of weight loss. Compared with the untreated mice, the SAMP8 mice treated with 3TC showed a tendency to increase their body weight with age. These results are consistent with those reported previously, suggesting that 3TC improves the health of the bodies of the mice.34 In order to objectively and accurately evaluate the effects of 3TC treatment on improving the aging characteristics of mice, we recorded the senescence scores during the treatment. Our results showed that with the increase of 3TC treatment time, SAMP8 mice showed a tendency to decelerate aging. Specifically, the treatment of 3TC played a significant role in improving the glossiness of skin, the corneal opacity and the ulcer of the cornea caused by aging in SAMP8 mice. These results suggest that the anti-senescence target of 3TC exists on the skin and the corneal tissues. Previous studies confirmed that L1 was used as a target for improving degenerative diseases caused by aging, while reverse transcriptase inhibitors including 3TC were used as transposable inhibitors of L1.13 To date, the molecular mechanism of the role of L1 in the nervous system and the possibility of treating nervous system-related diseases such as AD by regulating L1 have not been convincingly explained. Furthermore, studies have suggested that the genetic recombination in the APP gene may be a key event in the pathogenesis of sporadic AD, which is a type of dementia.15 neuronal gene recombination has not been reported, while its occurrence and DNA damages and RNA intermediates require reverse transcriptase activities of protein functions. Therefore, reverse transcriptase inhibitors such as 3TC may play a role in the prevention and treatment of AD which causes cognitive decline. This speculation was confirmed by our results of Morris water maze experiments investigating the effects of 3TC on cognitive ability in SAMP8 mice.
FIGURE 7 Experiments in vitro confirmed the key molecules of 3TC against neuronal degeneration. The 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 a comparison with the control group. Red asterisks represent a comparison with the FA group
Our results of the Morris water maze tests showed that after 5 days of swimming training, SAMP8 mice having orally taken 3TC were able to find the hidden platform faster than untreated SAMP8 mice. Considering that the mice treated with 3TC showed larger body weight, which may be related to the number of skeletal muscles, we recorded the swimming speed of each group of mice in order to eliminate the effect of muscle strength. Although the mice taking 3TC swam faster than other groups on the first day of swimming training, the difference did not persist in the next 4 days. Therefore, we rule out the influence of muscle strength on the results of the water maze tests. Similarly, the results of the space exploration experiment on the 6th day demonstrated that SAMP8 mice treated with 3TC showed a tendency to find the original platform more actively than untreated mice. These results suggest that 3TC treatment improves the cognitive and memory declines caused by aging in SAMP8 mice. To explore the potential molecular mechanisms of 3TC improving brain aging, we applied real-time quantitative PCR to measure the mRNA levels of candidate genes in the hippocampal and cortical tissues of each group of mice. Our results suggested that 3TC treatment inhibited L1 induced by aging in the brain and decreased the expression of genes related to IFN, SASP and AD, indicating that the signal pathways activated by aging were suppressed by 3TC treatment. Our results suggest that 3TC may enhance intelligence and aging phenotypes by inhibiting L1 transposition to ultimately improve the chronic inflammation in the brain caused by aging. We note that more studies are needed to provide strong support for these molecular mechanisms of improving brain ageing by 3TC treatment revealed in this study. At present, the research on the pharmacological mechanism of reverse transcriptase inhibitors on nerve cells is being carried out in our laboratory using in vitro aging glial and neuronal cell models.
Our results of HE and Nissl staining experiments indicated that 3TC remediated the neuron loss in the hippocampus caused by aging, ultimately ameliorating the neurodegeneration by rescuing neurons from death. Besides, we observed more apoptotic cells in the hippocampus of SAMP8 mice in comparison with SAMR1 mice. Our results further demonstrated that 3TC treatment significantly reduced the number of apoptotic cells, probably due to the direct effect of 3TC on the apoptotic pathway-related proteins or other upstream molecules.
Learning and memory are important cognitive abilities that involve a complex network of functional brain regions working together to manage and process information. Therefore, any compounds showing effects on the brain should be taken cautiously with their potential targets fully understood. The network pharmacology approach is a systematic analytical technology used to explore the interaction networks of multiple factors such as diseases, drugs, genes and protein targets. We conducted the network pharmacology analyses, including PPI and enrichment analyses, based on a total of 269 potential targets (with 32 core targets) of 3TC annotated using Pharmmapper and ChEMBL databases. Our results demonstrated that 3TC may play important roles in maintaining the normal functions of the nervous system through both the estrogen signaling and the PI3K/Akt and neuroactive ligand-receptor interaction signaling pathways, which further explained the effects of 3TC on protecting the normal nerve functions and inhibiting the neuronal cell death. Representative molecules in the relevant pathways were selected to carry out molecular docking and in vitro experiments to confirm the results of bioinformatics analysis. In order to provide more evidence to support these speculated molecular mechanisms of 3TC working synergistically with the nervous system, further studies are necessary to identify the exact components involved and regulated in these pathways.
As a part of antiretroviral therapy (ART), 3TC has been extensively studied in the past years and has been approved for the treatment of chronic hepatitis B virus (HBV).35 It must be noted that if 3TC is used to treat aging-related neurodegenerative lesions, it is necessary to consider its pharmacological toxicity due to the weak immune system in elderly populations and cancer patients. Studies have shown that common adverse reactions of 3TC include upper respiratory tract infection, nausea, vomiting, abdominal pain, and diarrhea. Therefore, the clinical application of 3TC should be extremely cautious. At present, 3TC can be used as a pharmacological tool to study molecular mechanisms but not as a putative therapy. In summary, our study has demonstrated for the first time that 3TC improves the cognitive functions of SAMP8 mice by reversing the aging of the brain, providing experimental evidence for the use of reverse transcriptase inhibitors as potential treatments for neurodegenerative diseases.
ACKNOWLEDGEMENT
This work was supported by the National Natural Science Foundation of China (81670942, 81470704 and 81972057).
CONFLICT OF INTEREST The authors declare that they have no conflict of interest.
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