Chitosan from crabs (Scylla serrata) Represses Hepato-renal Dysfunctions in Rats via Modulation of CD43 and p53 Expression in High Fat Diet-induced Hyperlipidemia

Hepato-renal dysfunctions associated with hyperlipidemia necessitates continuous search for natural remedies. This study thus, evaluated the effect of dietary chitosan on diet-induced hyperlipidemic rats. Thirty male Wistar rats (90 ± 5.2) g were randomly allotted into six (6) groups (n=5): Normal diet, High-fat diet (HFD), Normal diet + 5% chitosan. The three other groups received HFD, supplemented with 1%-, 3%-, and 5% of chitosan. The feeding lasted for 8 weeks, after which the rats were sacrificed. Liver and kidneys were harvested for Analyses. Hepatic alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activity, and renal biomarkers (ALT, AST, urea, and creatinine) were assayed spectrophotometrically. Additionally, expression of hepatic and renal CD43 and p53 was estimated immunohistochemically. Hyperlipidemia caused a significant (p<0.05) decrease in the hepatic (AST, ALT, and ALP) and renal (AST and ALT) activities, while renal urea and creatinine increased. Furthermore, the HFD group showed an elevated level of hepatic and renal CD43 while p53 expression decreased. However, groups supplemented with chitosan showed improved hepatic and renal biomarkers, as well as corrected the aberrations in the expressions of p53 and CD43. Conclusively, dietary chitosan could effectively improve kidney and liver functionality via abatement of inflammatory responses.

. Indeed, chronic renal failure parallels with the occurrence of premature atherosclerosis and cardiovascular comorbidities and mortalities (Vaziri, 2006). Clinical evidence also suggests that atherosclerosis caused due to HLP predates renal failure (Kaysen, 1994). Furthermore, HLP contributes to the onset of fatty liver disease and is prodromal to type  (Ugbaja et al., 2020). Therefore, the mechanism underlying HLP-induced liver and kidney damage might be linked to inflammatory pathways and oxidative stress (Napoli and Flores, 2017). CD43, (Sailophorin or Leukosialin) is one of the most common leukocyte transmembrane sialoglycoprotein that is expressed by inflammatory cells such as monocytes, neutrophils, macrophages, and T-lymphocytes with distinct physiologic functions such as differentiation, proliferation, and apoptosis. But its overexpression has also been implicated in different tumors of non-hematopoietic cells and immune-deficiency (Balikova et al., 2012). Apart from its roles in DNA repair, cell cycle arrest, apoptosis, and oncogene activation (Vousden, 2009), p53 has been shown to coordinate intermediary metabolism such as regulation of glycolysis and repression of lipogenesis via the inhibition of sterol regulatory binding protein-1 (SREBP-1).
This way, p53 regulates lipid anabolism by compensatory activation of lipid oxidation pathways (Napoli and Flores, 2017). Moreover, alterations of p53 expression has been shown to contribute to aging and associated diseases, cardio-metabolic disorders, atherosclerosis, and cellular hyper-proliferation (Minamino et al., 2009;Napoli and Flores, 2017).
Chitosan (CTS), a de-acetylated derivative of chitin, is a natural polymer of glucosamine derived from the cell walls of some fungi, and the exoskeleton of crustaceans including shrimps, crabs, lobsters, and prawns (Zhang et al., 2012;Ugbaja et al., 2020). The hypolipidemic effect of chitosan has been extensively explored by many scientists (Zhang et al., 2011;Selvasudha and Koumaravelou, 2017). Further, chitosan has attracted much awareness as a biomedical material of importance, due to its reported expansive biological activities, including, but not limited to antitumor (Kumar et al., 2004), immune-stimulating (Jeon et al., 2001), anti-allergic (Vo and Kim, 2014), cholesterol-lowering, and antiinflammatory activities (Chung et al., 2012), and free radical scavenging activities (Ugbaja et al., 2020). Despite the extensive studies carried out on the bioactivities of chitosan, there is a dearth of information on the effect of chitosan with respect to HLP-induced hepato-and renocellular dysfunctions. In addition, the effect of chitosan on hyperlipidemia-invoked dysregulation of p53 and CD43 expression has not been explored. We therefore, examine the effects of chitosan on the hepatic and renal biomarkers as well as the expressions of CD43 and p53 in hyperlipidemic rats in vivo.

Experimental Animals
The High-fat diet containing 50% maize, 10% soya, 15% groundnut cake, 10% fish meal, 8% Palm kernel cake, 5% soya oil and 0.5% of methionine, lysine, grower premix and salt was mixed with the dietary chitosan using a blender to ensure uniform mixing of the components.
Diets were compounded to include 1%, 3%, and 5% chitosan, pelletized and feed to the animals for 8 weeks (Ogungbemi et al., 2020). The animals' body weight was monitored throughout the experimental period.

Experimental design
The experimental animals were divided into six (6) groups, each group with five animals.

Sacrifice and collection of samples
After the stipulated weeks of treatment, the rats were weighed and sacrificed after an overnight fast under light anaesthesia. The liver and kidney were excised, washed in cold physiological saline and frozen until needed for analyses. The organs were fixed in 4% phosphate buffered formalin for immuno-histochemical analyses.

Biochemical Analyses
Alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine transaminase (ALT) activities urea and creatinine levels were estimated by standard procedures according to the manuals from Randox Diagnostic Kits (Crumlin, England, United Kingdom).

Immuno-histochemical analyses for CD43 and p53
Paraffin-embedded sections of the liver and kidney tissues were de-paraffinized and rehydrated

Statistical Analysis
Data are expressed as mean ± standard error of mean. Analyses was done using statistical package for social sciences (SPSS) version 20, the level of homogeneity among the results test groups, was done using one-way analysis of variance (ANOVA), with p<0.05 considered significant. Where heterogeneity occurred, the groups were separated using Duncan Multiple Range Test (DMRT). Graphs were plotted using GraphPad Prism (Version 5).
Immunohistochemistry quantification was done using ImageJ in triplicates.

Cumulative weight gain of the experimental animals
There was significant (p<0.05) increase in the body weight of the animals fed with HFD only when compared with those fed with the normal diet (Table 1). Nevertheless, there was progressive reduction in the body weight of the animals fed with HFD containing varying level of chitosan (1, 3, and 5% respectively). The reduction in the body weight appeared to be lowest in the group fed with HFD + 1% chitosan.

Effects of chitosan supplementation on renal biomarkers
The specific activities of AST and ALT, as well as the levels of urea and creatinine of rats maintained on HFD and/or chitosan is depicted in figure 1. There were significant (p<0.05) decrements in the activities of AST and ALT in the group fed with HFD alone when compared with the normal diet group. However, groups supplemented showed gradual improvement in the activities of these enzymes. Interestingly, there appeared to be a hormetic response in the chitosan-treated group, as the 1% chitosan-supplemented group showed more improved activities of AST and ALT. Further, there was no marked difference in the AST and ALT activities between the normal diet and normal diets ± 5% chitosan. Urea level increased significantly (p<0.05) in the untreated HFD group when compared with the normal diet group. Similarly, the renal creatinine level increased in the HFD only group relative to the control.
Nevertheless, the group fed with HFD + 5% chitosan showed a lowered urea and creatinine level when compared with the HFD control group. The normal diet + 5% chitosan group did not differ from the normal diet group for both the urea and creatinine levels.

Discussion
This study provides a potential mechanism underlining the protective effect of crab chitosan on liver and kidneys of hyperlipidemic rats in vivo. The role of hyperlipidemia in the induction of oxidative stress has been linked to excessive production of ROS (Ugbaja et al., 2020). Expectedly, the increased cumulative weight gain (table 1) observed in the HFD untreated group is consistent with other study (Xu et al., 2008). This might be associated with increased food intake (Ogungbemi et al., 2020) as the excessive calorie is accumulated by the animal in this group culminating in excessive weight gain and the attendant hyperlipidemia. However, following supplementation with the chitosan, the cumulative weight was normalized suggesting the ability of the animals to regulate their feed intake. It could also be due to feeling of satiation and satiety usually associated with fibre-rich diet (Dreher, 2015).
ALT and AST are cytosolic enzymes whose activities has been used as an index of hepatic and Taken together, this study shows that chitosan derived from crabs possesses hepato-renal protective effects against hyperlipidemia-invoke damages. This is mediated by modulation of kidney and liver biomarkers and down regulation of pro-inflammatory CD43 expression and upregulation of p53 in rats submitted to high-fat diets.