Anti-Aging Effect Of Chitosan Oligosaccharide On D-Galactose-Induced Subacute Aging in MiceⅡ
Apr 26, 2023
3. Discussion
It has been widely accepted that the D-gal-induced subacute aging mouse model used in our present study is a usually adopted aging model based on the metabolic theory of aging, the symptoms of which are similar to the natural aging process. D-gal is a normal nutrient that naturally exists in the body (21). As a certain dose of D-gal is injected into the mice within a period of time, the concentration of it in cells will be too high to be catalyzed by galactose oxidase to aldose and hydrogen peroxide finally generating superoxide anions (22). The oxidation in the body produces a large number of free radicals, which are beyond the body's scavenging capacity and lead to lipid peroxidation; meanwhile, the final decomposition products (such as MDA) can directly or indirectly combine with proteins nucleic acids, phospholipids, and other substances, not only destroying the chemical structure of intracellular life substances and disrupting cell function but also damaging normal tissue cells, as well as affecting the normal osmotic pressure, which further lead to metabolic disorders of vital organs and eventually organism aging (23,24). Thus, D-gal-induced subacute aging in mice has been chosen in our current study to investigate the possible anti-aging effects of COS and explore the underlying mechanism, In the present study, the results revealed that the model group mice had a significant difference in their daily behaviors compared with those of normal group mice. In addition, the spleen and thymus of model mice significantly atrophied, while the indices of their spleen and thymus were significantly lower than those of normal mice. Moreover, vitalities of antioxidant enzymes, such as GSH-Px, CAT, and SOD in the liver and kidney of model mice were reduced, while the opposite was true for the MDA level. These results are in line with previous studies (6,7,22-24), indicating that the in mice was successfully established in our present study.
Click Here To Get More Knowledge about Cistanche For Anti-aging Effects
With the increase of age, the immune system of the body suffers degenerative changes that not only decrease the immune response to a foreign specific antigen but also present with a general imbalance of immune function, which finally led to the occurrence of various diseases (25). Therefore the regulation of body immunity is one of the main methods in the anti-aging study. Additionally, the thymus and spleen are two important immune organs of the body, the organ indices of which can initially reflect the strength of a non-specific immune system, and which are also the preliminary indices to estimate the non-specific immune function of the body (26,27. The present results showed that compared with the normal group, the immune organs, such as the thymus and spleen of mice in the model group injected with D-gal for eight weeks, were obviously shrunk, and the thymus and spleen indices were distinctly decreased. When compared with the model group, although, for treatment, there was no obvious promotion impact to this D-gal-induced diminution of thymus and spleen indices, these two organs' indices in the COS low-middle-high dose groups gradually increased (especially for the spleen index), indicating that COS could improve the spleen and thymus quality indices of senile mice, inhibit the degeneration of the immune organs, and ultimately enhance the non-specific immunity to some degree. On the other hand, as one of the nonspecific immune factors of the body, the antibody system plays an important role in the body's immune response and immune adaptation, while immunoglobulin (lg) is a commonly used indicator of humoral immune status (28-30]. Serum lg includes lgG, IgM, IgA, IgD, and IgE, of which lgG and IgM are the main components of serum antibodies, as their content can reflect the immune response capacity and humoral immunity of the body (31]. The results in the present study showed that the serum levels of lgG in the model group mice were significantly lower than those in the normal group mice. Compared to those of the model group, for VE-treatment, there was also no marked promoting effect on theseD-gal-produced reduction of lgG and lgM levels in mice serum, while these serum levels of IgG' andlgM of senescent mice treated with COS increased significantly, indicating that COS could improve the immune response ability and enhance the humoral immune function of the body. In summary, COS has a good regulatory role on the immune function of D-gal-induced aging mice, which supposedly serves to delay aging.
In addition, the endogenous antioxidant enzyme system, mainly including SOD (an enzyme devoted to scavenging superoxide anion radicals and reducing the production of lipid peroxidation), CAT (an enzyme dedicated to scavenging H2O2 to protecting peroxidation of cell wall lipid and lipoproteins), and GSH-Px (an enzyme concentrated on catalyzing GSH into GSSG and stimulating the toxic peroxide reduction into non-toxic hydroxyl compounds), is an important defense system against free radical damage in the body, the main function of which is to maintain the homeostasis of ROS in the internal environment and to remove excessively high levels of ROS [4,5,32,33]. Numerous pieces of evidence exhibit that the excessive production of ROS in biological systems can cause oxidative damage to tissues, impair membrane functions, affect cellular metabolism, and passivate proteins and enzymes, especially in the organs with fast metabolic processes like the liver and kidney [34–36]. Consistent with these studies, we found that repeated D-gal-injection induced an observable decrease in the activities of these key antioxidant enzymes (SOD, CAT, and GSH-Px), and markedly aggravated lipid peroxidation, manifested as the accumulation of MDA, indicating that oxidative damages occurred in mice liver and kidney. Furthermore, the serum levels of ALT, AST, and ALP are widely used to evaluate liver functions, in which ALP plays a very important role in the dephosphorylation of protein enzymes, while ALT and AST are the earliest and most sensitive indicators of liver injury. Meanwhile, UA and CREA are the main indicators of renal function, which will be filtered out through the glomerular fluid into the bloodstream in large amounts when renal damage occurs during the aging process [37,38]. Accompanied by oxidative tissue damage of mice's liver and kidney, the serum levels of ALT, AST, and ALP, as well as CREA and UA in the model group mice, were signifificantly higher than those of normal group mice. However, COS, from shrimp shell chitosan by enzymatic hydrolysis, not only exhibited favorable antioxidant properties by enhancing the activities of these antioxidases and decreasing the level of MDA, showing the oppositional effect on D-gal-induced oxidative damage to mice liver and kidney, but also mitigated these exceptional rises of serum ALT, AST, ALP, CREA, and UA levels to an extent lower than the model group, indicating an important role in preserving the normal function of the liver and kidney. Meanwhile, VE also displayed, to a certain extent, an antioxidant effect mainly by intensifying the activities of SOD in mice's liver and kidney, as well as reducing the levels of MDA, to implement a degree of protective effects on mice hepatic and renal functions. Additionally, histological examination showed that the levels of inflflammatory cell infiltration and necrosis of hepatocytes, as well as the atrophy of the renal glomerulus were enhanced in the model group, whereas the application of COS and VE ameliorated these hepatic and renal structure damages induced by chronic administration with D-gal to a certain extent, and the histological status in the COS-H group closely resembled that of the normal group. These results of histological assessments corresponded well with the biochemical analysis.
To sum up, VE, the monomer of which is often used as the positive control for the studies of subacute aging in mice induced by D-gal, is a well-known fat-soluble vitamin with excellent antioxidant activity [37,39,40]. Given that VE showed no prominent promoting effect on this D-gal-produced reduction of immune function indicators of mice, it was reasonable to infer that the certain protective effect of VE on the liver and kidney of D-gal-induced aging mice might be mainly due to its anti-oxidation quality. Relatively, COS, derived from the shells of shrimp and crab shells in the ocean, possesses a variety of biological effects, such as the increasing effect on this D-gal-induced reduction of immune function indicators of mice, and D-gal-caused decreases in the activities of key antioxidant enzymes in mice liver and kidney [12,15–17]. To comprehensively conclude our results, it could be regarded that COS possessed hepatoprotective and renoprotective effects on D-gal-induced subacute aging mice to realize its anti-aging activity, which might be associated with the anti-oxidant capability of COS, as well as its good regulatory role on mice immune function to a larger extent.
4.1. Materials and Chemicals
study were of analytical grade.
4.2. Animals
lighting cycle during the experimental period of eight weeks. They could access to the standard pellet diet and drink water ad libitum throughout the study period.
4.3. Preparation of Subacute Aging Mouse Model and COS Treatment
After adaptation for one week, mice were randomly divided into six groups of eight mice each according to Table 3. In order to establish a natural aging model mouse, D-gal (250 mg/kg/day) was injected subcutaneously into the back of the neck of mice once a day for 56 consecutive days. While mice in the normal group received equal amounts of physiological saline once daily instead of D-gal. After subcutaneous injection of D-gal in mice for one hour, mice in normal and model groups were treated with distilled water by gavage (20 mL/kg/day); meanwhile, mice in the VE group were treated with VE by gavage (50 mg/kg/day) as a positive control according to recent reports [37,39,40]. Mice in COS-L, COS-M, and COS-H groups were treated with the sample solutions (three different doses of COS) per day by gavage, respectively, which were administered daily as the treatment schedule displayed in Table 3.
4.4. Daily Observation of Mice
During the experimental period, the body weight of mice was measured by an electronic balance once a week. In addition, the appetite, appearance, mental condition, and behavioral activity of mice in each group were observed and recorded per week, respectively.
4.5. Preparation of Mice and Sample Collection
calculated as the organ index. After that, the liver and kidney were stored immediately at −80 ◦C for the sequent biochemical analysis. There were no casual or obvious signs of toxicity throughout the course of the experiments and all of the mice involved survived.
4.6. Determination of Serum Indices
The serum was separated from the whole blood by centrifugation at 4000 rpm for 15 min at 4 ◦C and then stored at −80 ◦C for biological analysis. An automatic biochemistry analyzer (Mindary BS-480, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) was used to measure the levels of ALT, AST, ALP, CREA, UA, IgG, and IgM in the serum of mice according to the assay kit instructions.
4.7. Measurement of the Anti-Oxidation Activity in Mice Liver and Kidney
For biochemical analysis, organ tissues of each mouse including the liver and kidney were quickly removed to prepare 10% (w/v) homogenates in ice-cold 0.9% NaCl solution, followed by centrifugation at 3000 rpm for 15 min at 4 ◦C, and then the supernatant was kept frozen at −70 ◦C until biochemical analysis. The protein concentrations were measured by the bicinchoninic acid (BCA) method using bovine serum albumin as the standard. The activities of SOD, CAT, and GSH-Px, as well as the levels of MDA in the liver and kidney, were determined by the assay kits.
4.8. Histological Examination of Liver and Kidney
After sacrififice, organ tissue samples, including liver and kidney, were quickly stripped, fixed in 4% paraformaldehyde for 24 h, dehydrated in ascending grades of alcohol, then embedded in paraffin, and finally sectioned at a thickness of 5 µm. These samples were stained by HE for routine examination of these tissues.
4.9. Statistical Analysis
All quantitative data were presented as means ± SD. Experimental values were analyzed by one-way ANOVA. A value of p < 0.05 was considered to be statistically signifificant. All analyses were performed using Statistical Analysis Software (SPSS 17.0).
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Sergiev, P.V.; Dontsova, O.A.; Berezkin, G.V. Theories of aging: An Ever-Evolving Field. Acta Nat. 2015, 7, 9–18.
2. Ristow, M.; Schmeisser, S. Extending life span by increasing oxidative stress. Free Radic. Biol. Med. 2011, 51, 327–336. [CrossRef] [PubMed]
3. Kuilman, T.; Michaloglou, C.; Mooi, W.J.; Peeper, D.S. The essence of senescence. Genes Dev. 2010, 24, 2463–2479. [CrossRef] [PubMed]
4. Harman, D. Aging: A theory based on free radical and radiation chemistry. J. Gerontol. 1956, 11, 298–300. [CrossRef] [PubMed]
5. Schieber, M.; Chandel, N.S. ROS function in redox signaling and oxidative stress. Curr. Biol. 2014, 24, R453–R462. [CrossRef] [PubMed]
6. Li, S.S.; Liu, M.; Zhang, C.; Tian, C.Y.; Wang, X.X.; Song, X.L.; Jing, H.J.; Gao, Z.; Ren, Z.Z.; Liu, W.R.; et al. Purification, in vitro antioxidant and in vivo anti-aging activities of soluble polysaccharides by enzyme-assisted extraction from Agaricus bisporus. Int. J. Biol. Macromol. 2018, 109, 457–466. [CrossRef] [PubMed]
7. Zhao, H.J.; Li, J.; Zhang, J.J.; Wang, X.X.; Hao, L.; Jia, L. Purification, in vitro antioxidant and in vivo anti-aging activities of exopolysaccharides by Agrocybe cylindracea. Int. J. Biol. Macromol. 2017, 102, 351–357. [CrossRef] [PubMed]
8. Deursen, J.M. The role of senescent cells in aging. Nature 2014, 509, 439–446. [CrossRef] [PubMed]
9. Ginter, E.; Simko, V.; Panakova, V. Antioxidants in health and disease. Bratisl. Lek. Listy 2014, 115, 603–606. [CrossRef] [PubMed]
10. Jasiulionis, M.G. Abnormal epigenetic regulation of immune system during aging. Front. Immunol. 2018, 9, 197. [CrossRef] [PubMed]
11. Kim, S.K.; Rajapakse, N. Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review. Carbohydr. Polym. 2005, 62, 357–368. [CrossRef]
12. Zou, P.; Yang, X.; Wang, J.; Li, Y.F.; Yu, H.L.; Zhang, Y.X.; Liu, G.Y. Advances in characterization and biological activities of chitosan and chitosan oligosaccharides. Food Chem. 2016, 190, 1174–1181. [CrossRef] [PubMed]
Ask for more:
Email:wallence.suen@wecistanche.com whatsapp:+86 15292862950
