Views: 7 Author: Yu Qiu , Yan Su, Jia Song , Fangming Mou , Jia Gou , Xiaoqi Geng , Xinyue Li , Zhiqiang Nie , Jianxin Wang , Yu Zheng , Min Wang Publish Time: 2023-05-04 Origin: Food Science and Human Wellness Volume 12, Issue 6, November 2023, Pages 2417-2427
The pressure on people in modern society is increasing gradually, followed by escalating heavy mental burden, which makes depression a more common mental illness in urban life. Many scholars have put forward different perspectives on depression, and the monoamine neurotransmitter hypothesis is the most common one . Among these compounds, 5-hydroxytryptamine (5-HT) and dopamine (DA), two monoamines, play an important role in depression, and their metabolic abnormalities may lead to depression .
The chemical properties and biological activities of natural polysaccharides can be changed by molecular modification. Such process can not only enrich the structure types of polysaccharides but also provide more choices for the screening of high-activity or specific polysaccharides . It can better explain the structure-activity relationship of polysaccharides. Therefore, the chemical modification of natural products and the improvement of their activity have attracted the attention of many scholars.
Carboxymethyl is introduced into polysaccharides by chemical methods. Numerous studies have shown that carboxymethylated modification of polysaccharides can change the structure and the physicochemical properties, thus enhancing their pharmacological activity . At present, studies on carboxymethyl polysaccharides mainly focused on improving the antioxidant, antineoplastic, and immunomodulatory activities, while there are few reports on improving the antidepressant activity .
Marasmius androsaceus belongs to Basidiomycetes, Agaricales, and Tricholoma monogolicum Imai. This species is an edible fungus with antihypertensive, antiinflammatory, analgesic, antifatigue, antitumor, antidepression and other pharmacological effects . In our previous studies, the exopolysaccharide of M. androsaceus (MEPS) had been demonstrated to exhibit a good antidepressant effect . In addition, a purified fraction (MEPS2) was obtained from crude MEPS through the sequential purification with DEAE-52 and Sephadex G-100 column chromatography . The structure and anti-depressant mechanism of MEPS2 were analyzed. The results showed that MEPS2 exerted antidepressant-like effects that were mainly related to the catecholamine neurotransmitter system with specific sites at TH, D2DR, and CAMKII.
In this study, carboxymethyl polysaccharides of MEPS2 were prepared by carboxymethylated modification using chemical methods based on the previous work. Using the content of neurotransmitter in PC12 cell culture supernatant as an index, the carboxymethyl polysaccharide (C-MEPS2) with the best antidepressant effect was screened out. C-MEPS2 was examined by elemental analysis and scanning electron microscopy (SEM). The antidepressant mechanism of C-MEPS2 was further investigated at the cellular and animal levels. As mentioned above, their receptors in the metabolic pathways of related consumables and antagonists were used for the coadministration experiments. The aim of our study was to obtain polysaccharide derivatives with better antidepressant effect.
Fig. 1. SEM results of two kinds of polysaccharides. (A) MEPS2 (B) C-MEPS2.
PC12 cells are rat pheochromocytoma cells, which secrete a variety of neurotransmitters after differentiation, and have general characteristics similar to nerve cells. PC12 cells have been widely used in neuropharmacology and neurotoxicology research due to their advantages of easy acquisition, reproducibility, and fast proliferation. In previous studies, mice injected with doses of RES showed a reduction in body temperature and wandering rates, suggesting a good model for depression. PC-12H cells induced by RES were used to establish a depression model, and the antidepressant effect of C-MEPS2 was analyzed.
Treating PC12-H cells with an appropriate concentration of RES resulted in cell shrinkage, thinning of ganglia, and cell apoptosis. After a certain dose of C-MEPS2, the ganglion began to recover, the refractive index increased, the cell damage was weakened, and the cell activity was enhanced. Hoechst33258 can penetrate the cell membrane, and the fluorescence of apoptotic cells is more obvious than that of normal cells after staining. Both C-MEPS2 and MEPS2 can repair the nuclear damage caused by RES, and significantly reduce the fluorescence intensity of cells, among which C-MEPS2 plays a significant role. Disruption of the mitochondrial transmembrane potential is often associated with apoptosis, a phenomenon widely considered to be one of the earliest events in the apoptotic process. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form a red fluorescent polymer. JC-1 cannot accumulate in the mitochondrial matrix when the mitochondrial membrane potential is low. At this time, JC-1 is a monomer and can produce green fluorescence. Therefore, the change of JC-1 from red fluorescence to green fluorescence can be used as an indicator of early apoptosis. Both C-MEPS2 and MEPS2 can reduce the red-green fluorescence ratio and balance the mitochondrial membrane potential, and the effect of C-MEPS2 is better than that of MEPS2.
Fig. 2. The effect of MEPS2 on the damaged PC12-H cells. (A) Protective effect of MEPS2 (32.5, 65 and 130 μg/mL) to RES-induced PC12-H death via CCK8 (n = 6). (B) Effects of MEPS2 on the cell viability of the damaged PC12 cells. Data are shown as mean ± SD (n = 6). ###P < 0.001 compared with the control group. **P < 0.01, ***P < 0.001 compared with the model group.
Fig. 3. Fluorescence microscope results of two kinds of polysaccharides. (A) MEPS2 (130 μg/mL) and C-MEPS2 (130 μg/mL) restored the RES-induced nucleus morphological apoptotic alterations as analyzed via Hoechst 33258 staining (n = 6). (B) The blue fluorescence intensity. Data are expressed as mean ± SD (n = 6). ### P < 0.001 compared with the control, *** P < 0.001 compared with the RES-exposed cells. $$ P < 0.01 compared with the MEPS2-exposed cells.
Fig. 4. The results of JC-1 staining and fluorescence intensity about MEPS2 and C-MEPS2 (130 μg/mL). (A) Disruption of the mitochondrial membrane potential (MMP) caused by the 24 h exposure to RES was strongly restored by MEPS2 and C-MEPS2 (130 μg/mL) pretreatment as analyzed via JC-1 staining (n = 6). (B) Fluorescence intensity of the red/green ratio. Data are expressed as mean ± SD (n = 6). ###P < 0.001 compared with the control, *** P < 0.001 compared with the RES-exposed cells. $$ P < 0.01 compared with the MEPS2-exposed cells.
Fig. 5. Expression levels of the phosphorylation of TH, D2DR, and P-CAMKâ…¡ in the PC12-H; Expression of (A) TH, D2DR, and (B) P-CAMKâ…¡; (C) Relative contents of TH, D2DR, and P-CAMKâ…¡. Data are shown as mean ± SD (n = 6). ### P < 0.01 represented significant difference when compared with the control group; ** P<0.01, *** P < 0.001 represented significant difference when compared with the RES-exposed cells; $$ P < 0.01 compared with the MEPS2-exposed cells.
Fig. 6. The effects of MEPS2 and C-MEPS2 on the concentration of Ca2+ in the damaged cells. (A) Intracellular Ca2+ overload caused by RES was strongly reversed by MEPS2 (130 μg/mL) and C-MEPS2 (130 μg/mL) pretreatment as analyzed via Fluo-4-AM staining (n = 6). (B) Green fluorescence intensity. Data are expressed as mean ± SD (n = 6). ###P < 0.001 compared with the control, *** P < 0.001 compared with the RES-exposed cells.
Fig. 7. Study on the antidepression effect of C-MEPS2 on mice (n = 8). (A) FST (B) TST (C) AMPT antagonism (D) Raclopride antagonism. Data are expressed as mean ± SD (n = 8). # P < 0.005 compared with the control, ###P < 0.001 compared with the control, *** P < 0.001 compared with MEPS2(AB), C-MEPS2(CD); n.s. indicates no significant difference between treatments.
Depression is a common mental disorder that can directly affect normal life in severe cases. 5-hydroxytryptamine (5-HT) and dopamine (DA), two monoamines, play an important role in depression . In the regulation of 5-hydroxytryptamine synthesis and expression in the brain, spinal cord and gastrointestinal tract, microorganisms have a considerable influence on the 5-hydroxytryptamine biosynthesis and physiological changes in the host intestine . Based on previous studies , our studies showed that MEPS2 and C-MEPS2 mainly affect the synthesis and metabolism of catecholamines, such as dopamines, while dopamines have little effect on intestinal flora, or there is no strong evidence for a relationship between them.
Previous studies have found that polysaccharide extracted from the fermentation broth of M. androsaceus (MEPS2) have a good antidepressant effect . A number of researches have shown that chemical modification can change the chemical activity of the original polysaccharides. In this study, MEPS2 was modified to enhance its antidepressant effects and obtain a polysaccharide that was more effective against depression . Therefore, carboxymethyl polysaccharides were prepared under different conditions by NaOH-to-chloroacetic acid method, and the carboxymethyl polysaccharide (C-MEPS2) with the best antidepressant effect was screened by measuring the PC12-H cells in the supernatant of indicators. Therefore, C-MEPS2 was used for further research.
PC12 cells are rat pheochromocytoma cells that secrete various neurotransmitters after differentiation and have general characteristics similar to nerve cells . PC12 cells are widely used in neuropharmacology and neurotoxicology research due to their advantages, such as easy access, reproducibility, and rapid proliferation . In the authors’ previous studies, mice injected with a certain dose of RES showed reduced body temperature and the loitering rate, suggesting that was a good model of depression . RES was used to induce PC-12H cells to establish a depression model and analyze the antidepressant effects of C-MEPS2.
PC12-H cells were treated with RES at an appropriate concentration. The cells shrank, the ganglion became thinner, and the cell began to apoptosis. When given a certain dose of C-MEPS2, the ganglion began to recover, the refractive index became stronger, the cell damage was weakened, and the cell activity was enhanced. Hoechst33258 could penetrate the cell membrane, and the fluorescence of the apoptotic cells was more obvious after staining than that of normal cells . Both C-MEPS2 and MEPS2 could repair the nuclear damage caused by RES, significantly reducing the fluorescence intensity of the cells while C-MEPS2 had a significant effect. The destruction of the mitochondrial transmembrane potential is often associated with apoptosis, and this phenomenon is widely regarded as one of the earliest events during apoptosis. JC-1 is an ideal fluorescent probe widely used to detect the mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form a polymer that can produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot be aggregated in the matrix of mitochondria. At this time, JC-1 is a monomer and can produce green fluorescence. Therefore, the change in JC-1 from red fluorescence to green fluorescence can be used as an indicator of early apoptosis . C-MEPS2 and MEPS2 could reduce the red-green fluorescence ratio and balance the mitochondrial membrane potential, and C-MEPS2 had a better effect than MEPS2.
Introducing a certain amount of carboxymethyl groups into the polysaccharides can not only enhance the electronegativity and solubility of the polysaccharides in water to a certain extent but can also be especially beneficial to the full play of biological activity . In recent years, carboxymethyl polysaccharides has attracted extensive attention from many scholars. Xu et al. found that carboxymethylation reduced the molecular weight of polysaccharides, decreased the apparent viscosity, and increased the water solubility. These physicochemical changes may be due to the introduction of the carboxymethyl groups, which increased the electron cloud density and electron-absorbing activity of polysaccharides, thus enhancing the interaction with positively charged biomolecules . FT-IR results showed that the carboxymethyl characteristic absorption peak appeared, and hydroxyl strength was significantly enhanced compared with that of the original polysaccharide. This result indicated that the carboxymethyl polysaccharide was successfully modified. Before and after modification, the surface structures of the polysaccharides were considerable different, and the antidepressant activity of C-MEPS2 was significantly improved compared with MEPS2. We speculated that the improvement of polysaccharide activity after modification was related to the introduction of carboxymethyl.
Both MEPS2 and C-MEPS2 could reduce the immobility time of mice and have a certain antidepressant effect. However, the effect of C-MEPS2 was significantly higher than that of MEPS2. Previous studies have shown that the antidepressant effects of MEPS2 were related to the DA synthesis, TH, D2DR, and CAMKII play an important role in this process . C-MEPS2 significantly increased the contents of CA, DA, TH, and D2DR. The authors hypothesized that the antidepressant mechanism of C-MEPS2 was consistent with MEPS2 and further verified by the antagonist administration experiment. AMPT is a commonly used tyrosine hydroxylase inhibitor, and raclopride is a central DA D2 receptor specific antagonist . The antagonist administration experiment showed that coadministration of C-MEPS2 increased the inactivity time of mice compared with C-MEPS2 alone. This result further demonstrated that the antidepressant mechanism of C-MEPS2 was consistent with MEPS2 and related to the DA synthesis pathway and the D2DR. DA receptors are g-protein-coupled receptor families composed of seven transmembrane regions. According to the biochemical and pharmacological properties of DA receptors, these receptors can be divided into D1 and D2 receptors . In addition, intracellular Ca2+ accumulation or overload could lead to a range of neuronal damage, including neuronal degeneration, necrosis, and apoptosis . D2 receptors can suppress the Ca2+ current through coupling G0α in the midbrain DA neurons and activation of the electric current by D2 receptors. Other signal transduction pathway will be available, and DA receptors D2DR by Gi protein can increase the content of IP3. IP3 levels can increase the P-CAMKII expression and P-CAMKII to further promote the neurotransmitter release, thus improving depression (Fig. 8). A relevant study has measured the levels of metabolites (Tyr, DOPA, DA, and NE) associated with the catecholamine pathway in the hypothalamus in a mouse model of RES. MEPS2 was found that could increase the expression of TH, an enzyme that catalyzes amino acids and is a prerequisite compound for DA , thus increasing the content of catecholamine neurotransmitters. TH catalyzes Tyr entering the nerve endings to produce DAPA and then DA. NE in the cytoplasm of nerve endings is also affected by TH, and NE regulates the rate of TH production , thereby increasing the amount of neurotransmitter. Based on the above researches, we speculated that mechanism of action of C-MEPS2 is the same as that of MEPS2.
Fig. 8. Mechanism of antidepressant activity by C-MEPS2.
A derived polysaccharide was obtained by carboxymethylated modification. At the cellular level, the antidepressant effects of MEPS2 and the carboxymethyl polysaccharides were screened. MEPS2 and part of the carboxymethyl polysaccharides could increase the concentration of CA, NE, and DA, upregulate P-CAMKâ…¡ and D2DR expression, and lead to a substitution degree of 1.113 ± 0.009. MEPS2 and C-MEPS2 could stabilize the mitochondrial membrane potential and intracellular Ca2+ concentration to alleviate the apoptosis caused by RES. The effect of C-MEPS2 was superior to that of MEPS2, which was related to the introduction of carboxymethyl. In addition, the animal experiment further proved that C-MEPS2 exerted an antidepressant effect related to catecholamine synthesis, and the specific site were TH, D2DR and P-CAMKâ…¡. These data may provide new ideas for the clinical application of C-MEPS2 as an effective antidepressant product.
