*2.7. Expression of Caspase-3 Protein in Kidney*

During apoptosis, the caspase-3 protein was positively expressed, and the immunoreactive product was stained in renal tubules. The expression of the caspase-3 protein in the mouse kidney is shown in Figure 6. Apoptosis was observed in each group. Compared with the blank group, caspase-3 showed obvious positive expression in the NC group, and the positive cells were increasingly dense. The positive expression of UPE was significantly reduced in mice after UPE treatment, suggesting that UPE may have a protective effect on apoptosis. Image J software was used to analyze the staining situation, and it was found that compared with the BC group, the protein expression level of the NC group was significantly increased (*p* < 0.05), and the expression level of caspase-3 decreased significantly after treatment (*p* < 0.05), which further proved the improvement effect of UPE on apoptosis.

**Figure 6.** Effect of UPE on the expression of caspase-3 in kidney treated with D-gal. (**a**) The arrow in the image shows positive expression of caspase-3 protein and apoptosis (×400). (**b**) The expression of caspase-3. Data are presented as mean ± standard deviation (*n* = 9). \* *p* < 0.05 compared with BC group; # *p* < 0.05 compared with NC group. BC: blank control, NC: negative control, PC: positive control, LD: low-dose UPE (50 mg/kg), HD: high-dose UPE (300 mg/kg).

#### **3. Discussion**

The kidney is an important organ in the human body. Through filtration, the kidney educts the excess waste in the human body through urine, so as to maintain the metabolism of the human body and ensure the stability of the internal environment of the body. It has been reported that the production of large amounts of reactive oxygen species is the key pathogenic factor for a variety of kidney diseases [12]. In the event of oxidative stress, both renal tubules and vascular cells produce large amounts of reactive oxygen species, which attack renal cells and tissues and further cause renal damage [13]. In this study, in order to investigate the antioxidant ability of ulvan in vivo, we established a model of oxidative stress induced by D-gal to study the protective effect of UPE on kidney injury induced by oxidative stress.

Previous studies have shown that D-gal can lead to decreased indexes of kidney, thymus, brain, and other organs [14,15]. The results showed that UPE significantly improved the kidney atrophy induced by D-gal, which proved the protective effect of UPE on kidneys to a certain extent. This result was also confirmed in the H&E observation. D-gal-induced oxidative stress can cause damage to various tissues and organs. In mice treated with D-gal, capillary congestion of glomeruli and renal tubules would occur, and renal tubules would degenerate and die, leading to renal injury [16]. In our study, changes in renal structure were observed in mice. Compared with D-gal-treated mouse kidneys, UPE improved the pathological phenomena of glomerular atrophy and renal lumen obscuration, suggesting that UPE had a certain therapeutic effect on mouse kidney injury induced by D-gal treatment.

Excessive production of reactive oxygen species can cause oxidative damage to biological macromolecules such as proteins, nucleic acids, lipids, and DNA [17]. Lipid is the most common target of oxidative stress, and its oxidation product MDA can cause serious damage to mitochondrial respiratory chain complex and cell membrane, and it is widely used as a marker of lipid peroxidation [18]. Although 8-OHDG is not a specific marker of oxidative damage, ROS and hydroxyl radicals can attack DNA to produce 8-OHDG, which is the most commonly used biomarker of oxidative damage of DNA [19]. The carbonyl group is a key marker of protein oxidation, which is formed because reactive oxygen species directly attack the free amino group in amino acid molecules [20]. GSH is an important antioxidant and free radical scavenger in the body, which can react with H2O2 under the catalysis of GSH-Px to form GSSH, and remove peroxides and hydroxyl radicals produced by cellular respiration metabolism [15]. As an important component of the body's antioxidant defense system, SOD is the primary material for scavenging free radicals in the body, which can convert superoxide free radicals into hydrogen peroxide. GSH-Px protects cells from damage by scavenging lipid and hydrogen peroxides [21]. T-AOC is an evaluation index of comprehensive antioxidant capacity. The changes of oxidation products (MDA, 8-OHdG, protein carbonyl group, and GSH) and antioxidant enzyme activities (SOD, GSH-Px, and T-AOC) can be used to determine the oxidation level in the body. Through our study, it was found that the levels of MDA, 8-OHdG, and protein carbonyl in mice after UPE treatment were significantly reduced, suggesting that UPE has a significant therapeutic effect on oxidative injury. At the same time, we also found that UPE could significantly increase the activities of SOD, GSH-Px, and T-AOC in mice, which was consistent with the study of Liu et al., which has shown that polysaccharide extracted from *U. lactuca* could improve the antioxidant capacity of SAMP8 mice and also reduce the contents of inflammatory factors such as TNF-α, IL-6, and IFN-γ in mice [22]. These results provide evidence that UPE can be used to treat oxidative stress.

Kidney damage is followed by problems with kidney function. Scr, BUN, and Cys-C are commonly used to measure kidney health, and their concentrations are increased by kidney injury or renal failure [23]. The creatinine and urea in the blood are filtered mainly in the glomerulus, part of which is filtered out of the blood and the rest of which is reabsorbed. Serum Cys-C has a better effect in marking glomerular filtration level and can therefore be used as a marker of glomerular filtration rate [24]. In this study, after acute kidney injury, Scr, BUN, and Cys-C levels were significantly increased, indicating a sharp decrease in renal tubular filtration rate, which was consistent with previous studies [25]. Based on these conclusions, the contents of serum creatinine, blood urea nitrogen, and Cys-C decreased significantly after treatment with UPE and approached the normal level with the increase in the concentration, indicating that the filtration ability of mouse kidneys gradually recovered. Studies have shown that alginate oligosaccharides have an obvious protective effect on kidney injury induced by D-gal in mice, and the levels of BUN and Scr are improved after 4 weeks of treatment [26]. Kelp polysaccharide can reduce the content of Scr and BUN. This protective effect may be related to the anti-inflammatory and antioxidant effects of sulfated polysaccharides [27]. Therefore, it was speculated that the protective effect of UPE on the kidney might be related to its antioxidant capacity.

Oxidative stress can activate a variety of transcription factors, such as NF-κB, AP-1, and P53, which lead to the expression of inflammatory cytokines, anti-inflammatory molecules, and other genes [28]. TNF-α is a major inflammatory cytokine that can kill target cells, promote cell apoptosis, and participate in local inflammation and activation of endothelial cells. IL-6 is a type of interleukin that acts as a proinflammatory cytokine and an anti-inflammatory myosin. In our study, the levels of IL-6 and TNF-α in mice in the BC group were significantly higher than those in the control group, indicating a severe inflammatory response in mice. It may be caused by the production of proinflammatory mediators stimulated by oxidative stress. Although the content of IL-6 in mice was decreased after using UPE, the effect was not obvious, but the level of TNF-α in mice was decreased significantly (*p* < 0.05). Some studies have shown that *U. lactuca* polysaccharide can significantly reduce the serum levels of TNF-α and NO in breast cancer mice, indicating that UPE can regulate inflammatory response and reduce the level of inflammatory cytokines [29]. It has also been reported that *Gracilaria* extract achieves anti-inflammatory effects by reducing the levels of NO, IL-6, and TNF-α [30]. Purified kelp polysaccharide can reduce inflammatory reactions by inhibiting the activation of the TGF-β1-mediated inflammatory cytokine signaling pathway, downregulating the levels of TNF-α and IL-1β, etc. [31]. It was suggested that UPE might regulate the expression of cytokines at the transcriptional level [32].

Apoptosis is closely related to oxidative stress induced by ROS. Oxidative stress can mediate apoptosis by stimulating the synthesis of inflammatory factor TNF-α. TNF-α can activate caspase-8 after binding to surface receptors and then shear and activate caspase-3, leading to the production of apoptosis [33]. In addition, excessive ROS will directly attack the mitochondrial membrane, change the ratio of proapoptotic protein Bax and anti-apoptotic protein Bcl-2, mediate the release of cytochrome C, activate apoptotic factor caspase-3, and cause cell apoptosis [34]. Cascade activation of the cysteine protease family is an essential procedure in apoptosis, in which caspase-3 plays a key role [35]. In this study, the renal tubular epithelial cells of mice treated with D-gal showed a strong positive reaction, the expression of caspase-3 was enhanced, and abnormal renal apoptosis occurred. However, after UPE treatment, the apoptosis of renal cells was significantly reduced, which indicated that UPE had a certain protective effect on the kidney. It has been reported that *Enteromorpha prolifera* polysaccharides can regulate the mitochondrial apoptosis pathway and reduce cell damage [36]. Astragalus polysaccharides can also protect acute kidney injury by regulating oxidative stress and improving mitochondrial apoptosis signals [37]. These results indicate that UPE has a good effect on apoptosis induced by D-gal and provide a direction for the development of UPE in the future.

#### **4. Materials and Methods**

#### *4.1. Chemicals*

D-gal and ascorbic acid (VC) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Guangzhou, China). Assay kits for the measurements of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), malondialdehyde (MDA), total antioxidant capability (T-AOC), serum creatinine (Scr), blood urea nitrogen (BUN), serum cystatin C (Cys-c), 8-hydroxylated deoxyguanosine (8-OHdG), and protein carbonyl were purchased from Jiancheng Bioengineering Institute (Nanjing, China). ELISA detection kits for IL-6 and TNF-α were obtained from Dakewe Biotech Corporation (Beijing, China).
