3.1 Effect of LTA on body weight, OFT, LDT, and the structure of the hippocampal CA1 area of the brain in rats fed different protein diets
After 40 d of high protein feeding, the body weight was not significantly changed in any group compared to the P2HL group (P > 0.05, Fig. 1B). The average daily feed intake was significantly lower in the P3, P4, and P5 groups than in the P2 group (P < 0.05, Fig. 1C).
The frequency of open-field horizontal activity was significantly higher in the P3CK group than in the P2CK group (P < 0.05, Fig. 1D). However, the frequency of open-field vertical activity was not significantly different between P2CK and P3CK (P < 0.05, Fig. 1E). Additionally, the frequency of open-field horizontal activity was significantly lower in the P4CK and P5CK groups than in the P2CK group (P < 0.05, Fig. 1D). Similarly, the frequency of open-field vertical activity was significantly lower in the P4CK and P5CK groups than in the P2CK group (P < 0.05, Fig. 1E). ML and HL significantly increased open-field horizontal and vertical activity frequency under 20% and 40% protein diets (P < 0.05, Fig. 1D and E). All doses of LTA significantly increased the frequency of open-field horizontal and vertical activity under a 50% protein diet (P < 0.05, Fig. 1D and E). All doses of LTA showed no significant change in the frequency of open-field horizontal under the 30% protein diet. Interestingly, ML and HL significantly increased the frequency of open-field vertical under a 30% protein diet.
The number of visits to the bright box and the time entering the bright box were significantly higher in the P3CK group than in the P2CK group (P < 0.05, Fig. 1F and G). However, these were lower in the P4CK and P5CK groups than in the P2CK group (P < 0.05, Fig. 1F and G). ML and HL significantly increased the number of visits to the bright box under the 20%, 30%, 40%, and 50% protein diets (P < 0.05, Fig. 1F). All doses of LTA significantly increased the time entering the bright box under 20%, 40%, and 50% protein diets (P < 0.05, Fig. 1G).
The HE and NS results in the CA1 area of the rat hippocampus showed that compared with P2CK, the vertebral cells of the P4CK and P5CK groups were loosely and irregularly arranged (Fig. 1H and I). The number of neurons was reduced, or the shape changed. The nucleus appeared pyknotic and necrotic cells increased. Meanwhile, the Nissl staining revealed few, lightly stained or dissolved Nissl bodies, and the nucleolus was severely fragmented. This indicated weakened neuronal function. The CA1 area of rat hippocampi of the P3CK group was similar with that of the P2CK group. After 40 d of LTA intervention, compared with different HPD groups, the neuron damage in each LTA intervention group was reversed, cell morphology was clear, number of cells increased, number of necrotic cells decreased, and Nissl bodies increased.
Pathological changes of the liver can be reflected more intuitively by liver sections. Figure 2G shows that compared with the P2CK group, the liver histopathological analysis of the P3 groups showed no obvious signs of pathological changes. In the liver tissue sections of the P4CK and P5CK groups, varying degrees of liver cell granular degeneration, formation of fatty vacuoles of varying sizes, unclear liver cord structure, widening of hepatic sinusoids, and local hepatocyte necrosis accompanied by inflammatory cell infiltration were observed. After the intervention with each dose of L-theanine, lipid degeneration and inflammatory infiltration were relatively reduced, and liver structural damage was alleviated.
3.2 Effect of LTA on ALT, AST, T-SOD, CAT, MDA, CRP, and the structure of the liver
The content of ALT, AST, T-SOD, CAT, MDA, and CRP in liver are presented in Fig. 2. The contents of ALT (Fig. 2A), AST (Fig. 2B), CAT (Fig. 2C), T-SOD (Fig. 2D), and MDA (Fig. 2E) are no significantly difference in P3 groups than in P2 groups (P > 0.05). However, the content of CRP is significantly higher in P2CK and P3CK than in P2 and P3 LTA-treated groups (P < 0.05, Fig. 2F). Additionally, the content of ALT (Fig. 2A), AST (Fig. 3B), MDA (Fig. 2E), and CRP (Fig. 2F) are significantly higher in P4CK and P5CK than in P2CK (P < 0.05). Similarly, the content of CAT (Fig. 2C) and T-SOD (Fig. 2D) are significantly lower in P4CK and P5CK than in P2CK (P < 0.05). On the contrary, all dose of LTA significantly decreasing the content of ALT (Fig. 2A, P < 0.05), AST (Fig. 2B, P < 0.05), MDA (Fig. 2E, P < 0.05), and CRP (Fig. 2F, P < 0.05), except AST in P4LL. Similarly, all dose of LTA significantly increasing the content of CAT (Fig. 2C, P < 0.05) and T-SOD (Fig. 2D, P < 0.05).
3.3 Effect of LTA on the content of neurotransmitters in serum, brain, and liver in different levels of protein diet fed rat
The contents of DA, 5-HT, and NE in rat serum are presented in Fig. 3A, B, and C. The contents of DA, 5-HT, and NE were significantly higher in the P3CK group than in the P2CK group (P < 0.05, Fig. 3A). Meanwhile, the DA content was significantly lower in P5CK, and the contents of 5-HT and NE were significantly lower in P4CK and P5CK than P2CK (P < 0.05, Fig. 3A). After LTA intervention, the DA content was significantly higher in the P4ML and P4HL groups than in the P4CK group and significantly higher in the P5ML and P5HL groups than in the P5CK group. The content of 5-HT was significantly higher in the P3ML and P3HL groups than in the P2CK group, significantly higher in the P4HL group than in the P4CK group, and significantly higher in the P5LL, P4ML, and P5HL groups than in the P5CK group (P < 0.05, Fig. 3B). There was no significant change in NE content after LTA intervention (P > 0.05, Fig. 3C).
The levels of BDNF, ACh, GABA, and Glu in the rat brain are presented in Fig. 2D, E, F, and G. The levels of BDNF and ACh were significantly higher in the P3CK group than in the P2CK group (P < 0.05, Fig. 3D, E, and F). BDNF and ACh levels were significantly lower in the P4CK and P5CK groups than in the P2CK group (P < 0.05, Fig. 3D, E, and F). All doses of LTA significantly increased the content of BDNF in the 20%, 30%, 40%, and 50% protein diets (P < 0.05, Fig. 3D and G). Meanwhile, all doses of LTA significantly increased the content of Glu in the 40% and 50% protein diets (P < 0.05, Fig. 3E). However, HL significantly decreased the content of Glu in the 30% protein diet (P < 0.05, Fig. 3E). The effect of LTA significantly increased the content of ACh in the P3HL, P4ML, P4HL, and P5HL groups (P < 0.05, Fig. 3F). The levels of GABA were significantly lower in the P4CK and P5CK groups than in the P2CK group (P < 0.05, Fig. 3G). All doses of LTA significantly decreased the content of GABA in the 40% and 50% protein diets (P < 0.05, Fig. 3G).
ACh, GABA, and Glu contents in the rat liver are presented in Fig. 2H, I, and J. The contents of ACH and GABA, were significantly higher in the P3CK group than in the P2CK group (P < 0.05, Fig. 3I and J). The contents of Glu and ACh was significantly lower in the P5CK group than in the P2CK group (P < 0.05, Fig. 3H and I). ML and HL significantly increased the content of Glu and ACh in the 20% and 30% protein diets, respectively, and all doses of LTA significantly increased the content of Glu and ACh in the 40% and 50% protein diets (P < 0.05, Fig. 3I). All doses of LTA significantly decreased the content of GABA in the 40% and 50% protein diets (P < 0.05, Fig. 3J).
3.4 Proteomes and metabolites analysis to predict the mechanism by which LTA regulates neurotransmitters in rats fed HPD
By analyzing behavior, neurotransmitter content in tissues, the structure of the brain hippocampus and liver, and liver biochemical indicators, we chose P2CK, P2HL, P4CK, and P4HL to explore the effects of LTA on metabolites under a high-protein diet. The samples from the four groups were analyzed using LC-MS/MS.
We obtained volcano diagrams in the positive and negative ion modes using the PLS-DA data model to screen the metabolites with different levels between the groups, as shown in Fig. 4A-F. There were 45 differential metabolites in positive ion modes and 30 in negative ion modes between P2CK and P2HL (Fig. 4A and B, Supplementary Tables 2 and 3). We found that the differential metabolites of LTA that interfere with normal diets were mainly enriched in dopaminergic synapses, tyrosine metabolism, bile secretion, and other pathways (Fig. 4G). There were 93 differential metabolites in positive ion modes and 60 in negative ion modes between P4CK and P2CK (Fig. 4C and D, Supplementary Tables 4 and 5). We found that the differential metabolites of the high-protein diet were mainly enriched in dopaminergic synapses and tyrosine metabolic pathways. (Fig. 4H). There were 45 differential metabolites in positive ion modes and 42 in negative ion modes between P4HL and P4CK (Fig. 4E and F, Supplementary Tables 6 and 7). We found that the differential metabolites of LTA to the body were mainly enriched in bile secretion, alanine, aspartic acid, and Glu metabolism, and arachidonic acid (AA) metabolism pathways in the 40% protein diet. (Fig. 4I).
Proteomes results showed that there were 4415 proteins and annotated the functions of these proteins. Clusters of orthologous groups (COG) analysis showed that these proteins were mainly enriched in nuclear structure, lipid transport, and metabolism (Fig. 5A). There were 98 differentially expressed proteins (DEPs) between the P2CK and P2HL groups; 47 DEPs were upregulated, and 51 DEPs were downregulated (Fig. 5B). There were 189 most DEPs between the P4CK and P2CK groups; 112 DEPs were upregulated, and 77 DEPs were downregulated (Fig. 5B). There were 154 most DEPs between the P4HL and P4CK groups (Fig. 5B); 48 DEPs were upregulated, and 106 DEPs were downregulated. GO enrichment analysis was performed to understand the internal relations among DEPs and the relationship between enriched DEPs and neurotransmitter biosynthesis. Most DEPs are related to the inflammatory response, taurine biosynthesis process, L-cysteine catabolic process to taurine, and L-cysteine catabolic process to hypotaurine between P2CK and P2HL (Fig. 5C). Most DEPs were related to the cholesterol biosynthetic process and aspartate metabolic process between P4CK and P2CK (Fig. 5D). Most DEPs were related to the regulation of retrograde trans-synaptic signaling by endocannabinoids, regulation of the innate immune response, and pyrimidine nucleotide metabolic process between P4HL and P4CK (Fig. 5E). First, we found one of the four DEPs related to neurotransmitters in P2HL vs. P2CK, namely PF4 (Supplementary Table 8). Notably, PF4 is a specific protein that promotes thrombosis. Eight DEPs showed opposite trends in their expression in P4CK vs. P2CK and P4HL vs. P4CK (Supplementary Table 9). One of the eight DEPs were related to neurotransmitters, namely ABHD12. ABHD12 exists in the brain and immune system of mammals and is closely related to neurodegenerative diseases [16]. It is also involved in 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (AEA) metabolism [25].
3.5 Western blot analysis of the effect of LTA on key proteins in the predicted pathways in HPD fed rat livers
The grayscale band intensities of GNAI2, ABHD12, AC, PF4, and PKA were showed in Fig. 6A, shown as a histogram. Compared to the P2CK group, the expression level of GNAI2 was significantly upregulated in the P2HL group (Fig. 6). However, our study showed that compared to the P2CK group, the expression levels of ABHD12, AC, and PKA were not significantly regulated in P2HL, indicating a limited effect of LTA on the AC/PKA signaling pathway via GANI2 on a normal diet. As shown in Fig. 6A, GNAI expression levels were significantly changed in the P4CK and P4HL groups. Compared to the P2CK and P2HL groups, the expression levels of ABHD12, AC, PKA, and PF4 were significantly changed in the P4CK group. Furthermore, compared to the P4CK group, the expression levels of ABHD12, AC, and PKA were significantly downregulated, and the expression level of PF4 was significantly upregulated in the P4HL group, indicating that a high-protein diet affects neurotransmitter metabolism by changing the expression of GNAI2 and ABHD12. Therefore, we speculated that LTA affects neurotransmitter metabolism by changing the expression of GNAI2 and ABHD12 and by acting on the AC-PKA signaling pathway.