Regulation of Laminaria Polysaccharides with Different Degrees of Sulfation during the Growth of Calcium Oxalate Crystals and their Protective Effects on Renal Epithelial Cells

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (–OSO3–) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca2+ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of –OSO3−, that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high –OSO3− contents are potential drugs for effectively inhibiting the formation of CaOx stones.


Introduction
The prevalence and incidence of kidney stones are on the rise globally, and no effective drug to treat or prevent the disease is currently available [1]. Calcium oxalate (CaOx) is the main inorganic component of kidney stones, accounting for about 70%-80% [2]. CaOx in kidney stones mainly exists in the form of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) [3]. COM is the most stable phase of thermodynamics and the most common form of stones. The incidence of stones caused by COM is twice that caused by COD [4]. Compared with COD, COM has a greater affinity for renal tubular cells [5] and is more difficult to be excreted in the urine. Therefore, COM in the body is more likely to induce kidney stone formation than COD crystals.
Oxidative stress-induced damage to renal tubular epithelial cells and decrease in inhibitor molecules in urine or in their activities are the important reasons for the formation of CaOx stones [6,7]. Increasing the concentrations and/or activities of inhibitors in the urine, removing excess free radicals in the body, and protecting renal tubular epithelial cells from oxidative damage may inhibit the formation and recurrence of kidney stones.
The chemical structures of plant polysaccharides are very similar to the structure of CaOx crystal growth inhibitor glucosamine (GAGs), which is commonly found in urine. Plant polysaccharides contain a large number of anionic groups, such as sulfate groups (-OSO 3 -) and carboxyl groups (-COO -), and can thus be used to inhibit the formation of CaOx kidney stones. Huang et al. [8] showed that plant polysaccharides can inhibit the growth and aggregation of COM and induce the formation of COD. Gangu et al. [9] found that Gum Arabic (GA) can regulate the phase and morphology of CaOx, promote the formation of COD crystals, and inhibit the growth of COM crystals.
High concentrations of oxalic acid and CaOx or CaP crystals activate NADPH oxidase, produce excessive reactive oxygen species (ROS), and induce the inflammation and oxidative damage of renal epithelial cells, leading to the death of renal epithelial cells [10]. Tubular epithelial cell injury can induce the high expression of cell surface adhesion molecules, such as hyaluronic acid, osteopontin (OPN), and CD44, resulting in the accumulation of CaOx crystals in the kidney [11,12]. Xi et al. [13] showed that high concentrations of calcium stimulate the attachment of CaOx crystals to rat renal tubular epithelial cells by inducing OPN expression, leading to the deposition of crystals in the kidney and the formation of CaOx stones. Plant polysaccharides can protect renal epithelial cells from oxidative damage by inhibiting the adhesion of CaOx crystals to cells. For example, Zhao et al. [14] found that tea polysaccharides can effectively protect HK-2 cells from COM damage, thus increasing cell viability, restoring cell morphology, increasing lysosome integrity, and reducing crystal adhesion. These features inhibit the production of kidney stones.
Plant polysaccharides not only effectively remove superoxide and various free radicals, such as hydroxyl, DPPH, and ABTS, but also reduce the level of lipid peroxidation, are less toxic, and have a lower number of side effects on the body. The biological activity of plant polysaccharides is not only related to its monosaccharide composition and molecular weight but also closely related to the content of active groups (such as -OSO 3 − and -COOH) in polysaccharide molecules. These active groups reduce the rate of negative charge loss on cell surfaces and repair the charge barrier. Chen et al. [15] obtained three types of sulfated polysaccharides with a sulfated contents of 9.11%, 10.33%, and 21.44% from Ganoderma atrum. The scavenging ability of these components on DPPH free radicals is positively correlated with their -OSO 3 contents, that is, polysaccharides with high -OSO 3 − contents are more resistant to oxidation. Chen et al. [16] modified Momordica charantia polysaccharide (MCP) by sulfation to obtain sulfated Momordica charantia polysaccharide (S-MCP) with a substitution degree of 0.45. S-MCP has a greater scavenging ability for superoxide anions than MCP. Lu et al. [17] modified chitooligosaccharides (COS) through sulfation to prepare COS-SI and COS-SII with substitution degrees of 0.8 and 1.9, respectively. Their protective effects on H 2 O 2 -induced MIN6 cell damage had the order COS-SII > COS-SI > COS, indicating that increasing the substitution degree of -OSO 3 − in polysaccharides prevent H 2 O 2 -induced oxidative damage in MIN6 cells.
Laminaria is an important economic seaweed and is widely found in the seas of China, Japan, and Korea. It is also the favorite food of some Asians. In the past thousand years, the Chinese has used it as a traditional medicine to treat edema disease [18]. Laminaria polysaccharide (LP) is the main active component of Laminaria. The antioxidant activity of LP was positively correlated with the -OSO 3 content of polysaccharide. For example, Wang et al. [19] obtained F1, F2, and F3 components with -OSO 3 − contents of 23.3%, 36.41%, and 36.67% from Laminaria. The scavenging ability of the components on superoxide radicals had the following order: F3 > F2 > F1, which indicates that the strength of the antioxidant activity of the polysaccharides increases with -OSO 3 content. Wang et al. [20] extracted fucoidan (FPS) with a -OSO 3 content of 27.56% from Laminaria and sulfated FPS to obtain SO 3 -FPS with a -OSO 3 content of 36.63%. The scavenging ability of SO 3 -FPS on hydroxyl radicals was significantly stronger than that of FPS.
However, the -OSO 3 content of polysaccharide has a great relationship with its producing area and extraction method [21][22][23]. For example, Yoon et al. [21] extracted three polysaccharide components (S1, S2, and S3) from Laminaria cichorioides in the East Sea of Korea, and the -OSO 3 content in S1, S2, and S3 were 0.09%, 2.19%, and 1.38%, respectively. Cui et al. [22] extracted Laminaria japonica polysaccharide (LJPA-P) from Laminaria japonica collected from Qingdao, Shandong, China, by a joint extraction of hot water and citric acid, and the -OSO 3 content of LJPA-P was 4%. However, the polysaccharides isolated and purified by LJPA-P (LJPA-P1 and LJPA-P2) could not even detect the -OSO 3 content. Saha et al. [23] collected crude Laminaria angustata polysaccharide (WEP) from the Okha coast of Gujarat, India. The -OSO 3 content of WEP was 3% and purified Laminaria angustata polysaccharide (F2) with a -OSO 3 content of 4.2%. These polysaccharides with low -OSO 3 content need to be sulfated to enhance their biological activity.
In this study, LP0 was sulfated to different degrees, and four kinds of SLPs (SLP1-SLP4) with -OSO 3 − contents of 8.58%, 15.1%, 22.8%, and 31.3% were obtained. The antioxidant activities, the ability to regulate CaOx crystals growth, and protective effects on renal epithelial cells of these SLPs were studied. The SLPs offer development prospects for drugs used in preventing and treating CaOx kidney stones.  [24]. The details are as follows: 10 mg of LP0 polysaccharide was added to a 121°C sealed container containing 2.5 mol/L trifluoroacetic acid (TFA, 2 mL) for 90 min. The solution was concentrated to dryness under reduced pressure; then, the TFA was removed with MeOH to a neutral solution and concentrated to dryness under reduced pressure. The residue was dissolved in 2 mol/L NH 4 OH (1 mL) and 1 mol/L fresh NaBD 4 (1 mL).

Materials and Methods
The reaction was carried out at room temperature for 2.5 h and stirring at room temperature. Then, two drops of acetic acid were added to decompose excess NaBD 4 until no bubbles producing. The solution was concentrated to dryness under reduced pressure. The filtrate was added MeOH to remove boric acid and dried in vacuo. Add 1 mL acetic anhydride and acetylate at 100°C for 2.5 h. The acetylated product was extracted with dichloromethane. The organic layer was washed with distilled water, dried, and analyzed by GC-MS. The HP-5MS capillary column (15 m × 250 μm × 0:25 μm) was programmed and the temperature was raised from 135°C to 180°C at 0.5°C/min, then to 190°C at 10°C/min and up to 310°C at 40°C/min. Helium acts as carrier gas, column flow rate 0.6 mL/min. The acetylated product was identified by debris ions in GC-MS and relative retention times in GC. The structure is identified by peaks and assessed by peak area. Standard monosaccharides (rhamnose, arabinose, fucose, sucrose, maltose, glucose, raffinose, fructose, and galactose) are used as references.

Sulfation and Characterization of LP0
2.4.1. Sulfation of LP0. According to the reference [25,26], the specific steps are as follows: accurately weigh 500 mg of LP0 into a 250 mL Erlenmeyer flask, add 30 mL of dimethyl sulfoxide, and stir at room temperature for 1 hour to completely dissolve the polysaccharide. Refer to Table 1 to add sulfur trioxide pyridine complex and 5 mL of formamide (sulfur trioxide-pyridine complex was dissolved in formamide in advance to make it transparent), and reacted for a certain period of time (0.5, 2, and 8 h) in a water bath. After the reaction, the sample was cooled to room temperature with ice water, neutralized to pH = 7:0 with 2.0 mol/L NaOH solution, and then transferred to a dialysis bag (Mw = 500 Da) for dialysis for 4 days, until the solution in the dialysis bag changed from light yellow to transparent. After dialysis, it is evaporated and concentrated to 3 mL at 50°C. The polysaccharide is precipitated with absolute ethanol. After standing overnight, it is centrifuged and dried to obtain sulfated polysaccharides SLP1, SLP2, SLP3, and SLP4 containing different -OSO 3 content.

Detection of -OSO 3 -Content in Polysaccharides.
The BaCl 2 -gelatin turbidimetric method was used to determine the -OSO 3 content of each SLPs [27], and K 2 SO 4 was used as a standard to draw a standard working curve. The regression equation was as follows: Y = 4:72803X + 0:01723, R 2 = 0:99498, n = 11.
2.4.3. FT-IR Spectrum of SLPs. Take 2.0 mg dried polysaccharide sample and 200 mg KBr, grind it to powder with an agate mortar, press the tablet, and scan in the wave number range of 4000-400 cm -1 . 13 C NMR Spectra of SLPs. According to the reference [28], weigh 20 mg dried polysaccharide sample, 3 Oxidative Medicine and Cellular Longevity dissolve it in a nuclear magnetic tube containing 0.55 mL of deuterium oxide (D 2 O). After being completely dissolved, it was placed in the magnetic field of a nuclear magnetic resonance spectrometer for detection.

The Regulation Effect of Different SLPs on the Growth of CaOx Crystals
(1) CaOx Crystal Synthesis. Add 20 mL CaCl 2 solution with a concentration of 22 mmol/L to a set of beakers, then add a certain amount of SLPs with different sulfation degrees, and add distilled water to make up to 24 mL. It is placed in a 37°C constant temperature water bath and magnetically stir for 5 minutes. Add 20 mL of 22 mmol/L Na 2 Ox solution to make the final volume of the system 44 mL. At this time, cðCa 2+ Þ = cðOx 2− Þ = 10 mmol/L in the solution, and the final polysaccharide concentration is 0.4 g/L, 0.8 g/L, 1.2 g/L, 1.6 g/L, and 2.4 g/L. The solution was stirred in a constant temperature water bath for 10 min, then transferred to a constant temperature water bath at 37°C and allowed to stand for 2 h, and centrifuged. The bottom CaOx precipitate obtained after centrifugation is dried in an oven. The soluble Ca 2+ ions concentration in the supernatant of the solution is measured by the ICP method. The bottom CaOx precipitate is dried in a dryer and weighed. After weighing, XRD, FT-IR, Zeta potential, and SEM tests are performed (2) XRD Detection. Take a certain amount of crystals and grind them into powder in a mortar for detection. The relative percentages of COM and COD in CaOx are calculated by the K value method according to the XRD spectrum In the formula, I COM and I COD are the intensities of the main diffraction peak ( 101) crystal plane (d = 0:593 nm) of COM and the main diffraction peak (200) crystal plane (d = 0:618 nm) of COD, respectively.   22.8% (SLP3), and 31.3% (SLP4) was added to react with 200 μg/mL COD for 15 min; then, it was added to normal cells and incubated for 12 h. After reaching the action time, add 10 μL of CCK-8 reagent to each well, incubate for 2 h at 37°C, measure the absorbance (A) at 450 nm with a microplate reader, and measure the cells under each condition in parallel in 5 replicate wells. Calculate the average value of A to test the protective ability of polysaccharides.
The concentration of polysaccharide in cell experiment was controlled by the following methods: serum-free medium was used to prepare a polysaccharide solution with a higher concentration (500 μg/mL), and then, a certain volume of the above polysaccharide solution is taken according to the target concentration (such as 20, 40, or 80 μg/mL) of the experiment, combined with the amount of other reagents added in the experiment, and finally diluted to the same volume with serum-free medium.

Cytotoxicity Detection of Polysaccharides.
Inoculate the cell suspension (density 1:0 × 10 5 cells/mL) in a 96-well plate, 100 μL per well, and incubate for 24 h in an incubator at 37°C, 5% CO 2 , and saturated humidity to make the cells converge into a monolayer. The cells were divided into two groups: (a) normal control group: only serum-free medium  2.8. Statistical Analysis. Experimental data were expressed by mean ± standard deviation ( x ± SD). The experimental results were statistically analyzed by the IBM SPSS Statistics 26 software, and the differences between the means of each experimental group and the control group were analyzed by Tukey. P < 0:05 indicates a significant difference; P < 0:01 indicates a very significant difference. P > 0:05 indicates no significant difference.

Measurement of LP0 Average Molecular Weight and
Monosaccharide Composition. The average molecular weight of LP0 is determined to be 1788 ± 78 Da by the Ubbelohde viscosity method. Figures 1(a) and 1(b) present the GC-MS results of standard monosaccharides and LP0. The majority of LP0 is composed of maltose and glucose with traces of sucrose, fructose, and raffinose. The mass ratio of maltose to glucose to raffinose to fructose to sucrose is 12.49 : 1.42 : 0.09 : 0.08 : 0.03.
As shown in Table 1, the -OSO 3 contents of the polysaccharides increase with reaction time, reactant concentration, and reaction temperature. However, SLP4 with an -OSO 3 content of as high as 31.3% can be prepared only by comprehensively controlling the three reaction conditions. Figure 1(c) shows the FT-IR spectra of SLPs with different -OSO 3 contents. The broad absorption peak around 3385 cm −1 is the stretching vibration of O-H. The peak at 2931 cm −1 is the -CH stretching vibration of the -CH 3 or -CH 2 group. The peak at 1640 cm −1 is the symmetric or asymmetric stretching vibration of C=O. The peak at 1024 cm −1 is the stretching vibration of C-O. The infrared characteristic absorption peaks of SLPs with different -OSO 3 contents are shown in Table 2.

FT-IR Spectrum Analysis of Different SLPs.
Compared with LP0, SLPs have two new peaks at 1259 and 820 cm −1 , which are the asymmetric stretching vibration of S=O and stretching vibration of C-O-S, respectively [29]. The intensities of the two peaks gradually increase with -OSO 3 content in the polysaccharides (Figure 1(d)), that is, LP0 < SLP1 < SLP2 < SLP3 < SLP4. This result indicates successful sulfation modification.  (Table 3) [31].
A new strong signal peak at δ 4.22 ppm was detected in the 1 H NMR spectrum of SLP2 after sulfation modification. The appearance of the peak is attributable to the change of chemical shift caused by the introduction of the -OSO 3    Oxidative Medicine and Cellular Longevity group after sulfation [32]. The signal peak at δ 3.96 ppm shifts to δ 4.22 ppm, indicating that the hydroxyl group of SLP2 is sulfated. The signal peak of SLP2 at δ 1.10 ppm is significantly enhanced because of the substitution of the hydroxyl group by the -OSO 3 group after the sulfation of the polysaccharide.
The 13 C NMR spectrum of SLP2 has the following changes relative to that of LP0: (1) The high field signal peak of SLP2 at δ 71.46 ppm is weakened, and a new peak appears at δ 76.20 ppm. The reason is the movement of carbon directly attached to the electron-withdrawing group (-OSO 3 group) after sulfation to a lower field position [34]. This movement indicates that the hydroxyl group at C-2 is sulfated (2) SLP2 shows signal peak splitting at δ 95-100 ppm.
The functionalization of the -OH group on C-2 causes the signal peak of C-1 to split, and this split has a good correlation with the substitution degree of the C-2 atom [35], which further shows that the hydroxyl group on C-2 is sulfated The new signal peak of SLP2 at δ 66.93 ppm is attributable to O-6 substituted carbon, indicating that O-6 is sulfated [36]. The peak of SLP2 at δ 16.77 ppm is significantly enhanced, indicating that the hydroxyl group at the C-6 position in the SLPs is replaced by -OSO 3 -According to the above analysis and reference [37], the -OH on C2, C4, and C6 of the original LP0 are replaced by -OSO 3 after the sulfation reaction (Figure 1(i)).  (Table 4), indicating that only the COM crystal is formed [38,39]. SLP2, SLP3, and SLP4 mainly induce the formation of COM at low concentrations and also induce COD crystal formation at high concentrations. The FT-IR spectrum of COD differs from that of COM. COD crystal has only one strong and broad absorption peak in the 3000-3600 cm −1 region, and ν as (COO − ) and ν s (COO − ) are at 1646 and 1330 cm −1 , respectively (Table 4) [38,39].

SLPs Regulate
Comprehensive XRD and FT-IR results show that the ability of SLPs to induce COD is positively correlated with the -OSO 3 − content of polysaccharides. Increase in the -OSO 3 − content or concentration of polysaccharides inhibits the formation of COM and induces the formation of COD. Figure 4(a) shows the SEM images of each SLP that induces the formation of CaOx crystals at 1.6 g/L. The crystals formed in the blank group have a strong three-dimensional effect, and serious aggregation phenomena occur. Compared with the crystals in the blank group, the crystals regulated by LP0 and SLP1 gradually transform into lamellae, but the degree of aggregation is weakened. XRD results show that the crystals formed in the blank, LP0, and SLP1 groups are COM crystals.

SEM Observation of CaOx Crystal.
COD crystals appear in SLP2-regulated crystals (approximately 27.7%, Figure 2(f)). Crystal size regulated by SLP3 and SLP4 is further reduced, the degree of aggregation between the crystals is further reduced, and the percentage of tetragonal bipyramid COD crystals is significantly

Detection of Soluble Ca 2+ Ion Concentration and CaOx
Precipitation in the System. The soluble Ca 2+ ion concentration in each SLPs system at 1.6 g/L was detected by ICP, and the mass of each CaOx crystal formed was weighed. As the -OSO 3 contents of the SLPs increase, the concentration of soluble Ca 2+ ions in the supernatant gradually increases (Figure 4(b)), whereas the amount of CaOx precipitates gradually decreases (Figure 4(c)).
For example, the masses of CaOx precipitates induced by LP0 and SLP4 are 63.5 and 66.5 mg, respectively, and respectively. This is because COD has one more water molecule than COM, which increases its relative molecular mass, so the mass of the precipitate increases with the same amount of substance (Table 5).
3.3.5. Zeta Potential of CaOx Crystal. Zeta potential can be used in measuring the amount of repulsive force between crystals. When the absolute value of the zeta potential increases, the strength of the repulsive force between crystals and the dispersion of crystals in a solution increases, and the aggregation of crystals is inhibited [40]. Figure 4(d) shows the absolute value of the Zeta potential of CaOx crystals produced by the regulation of five different SLPs with different -OSO 3 − contents. The absolute value of the zeta potential of the CaOx crystals produced by the regulation gradually increases. At the same concentration, as the -OSO 3 − content of polysaccharide increases, the absolute value of the crystal's Zeta potential gradually increases. For example, when the polysaccharide concentration is 2.4 g/L, the absolute values of the zeta potential of the CaOx crystals generated have the following order: LP0 (4.67 mV) < SLP1 (11.2 mV) < SLP2 (15.1 mV) < SLP3 (17.1 mV) < SLP4 (26.5 mV).

In Vitro Antioxidant Capacity of SLPs with Different
On the basis of the tested ability of SLPs with different -OSO 3 contents to scavenge hydroxyl (•OH) free radicals and DPPH free radicals, their in vitro antioxidant activities were compared. The -OSO 3 group in SLPs can inhibit the generation of hydroxyl radicals by chelating Fe 2+ [41]. In addition, when SLPs interact with DPPH, they transfer electrons or hydrogen atoms to DPPH, thereby neutralizing its free radicals [42]. Figures 5(a) and 5(b) shows the ability of SLPs with different -OSO 3 content to scavenge •OH radicals and DPPH radicals and their concentration effects. As the concentration of polysaccharides increases, the ability to scavenge •OH free radicals and DPPH free radicals increases, that is, the antioxidant capacities of polysaccharides are concentration dependent. Notably, a polysaccharide concentration of 1.0 mg/mL is a turning point for improving the speed of scavenging free radicals. When the polysaccharide concentration is less than 1.0 mg/mL, the ability of polysaccharides to scavenge free radicals increases rapidly with polysaccharide concentration. When the polysaccharide concentration is greater than 1.0 mg/mL, the ability of polysaccharides to scavenge free radicals increases slowly with increasing polysaccharide concentration.
At the same concentration, as the -OSO 3 contents of polysaccharides increase, the ability of the polysaccharides to scavenge free radicals increases, that is, the antioxidant capacities of SLPs are positively correlated with the content of -OSO 3 in SLPs. In •OH free radicals, SLP3 and SLP4 have a significantly stronger free radical scavenging ability 11 Oxidative Medicine and Cellular Longevity than other polysaccharides. For DPPH free radicals, SLP4 with a high -OSO 3 content has a significantly stronger free radical scavenging ability than other polysaccharides. decreases from 100:00% ± 1:27% to 54:84% ± 0:85%, indicating that COD crystals have obvious damage to cells, and this damage was moderate, which was convenient to carry out the protection experiment of polysaccharide [14,43]. The five polysaccharides used three concentrations of 20, 40, and 80 μg/mL to protect HK-2 cells. Cell viability test results show that cell viability increases compared with that in the nano-COD damage group because of the protection of HK-2 cells by the polysaccharides at each concentration,

13
Oxidative Medicine and Cellular Longevity and each polysaccharide had the best protective effect at 80 μg/mL.
The protective effect of each SLP on HK-2 cells was compared at the same concentration ( Figure 5(c)), and the results show that the degree of protection against nano-COD crystal damage in the HK-2 cells increases with the polysaccharide -OSO 3 content. At the best protective concentration (80 μg/mL), the cell viability of the five polysaccharide protection groups is 61.24% (LP0), 61.54% (SLP1), 67.87% (SLP2), 91.30% (SLP3), and 95.31% (SLP4).
3.5.2. Cytotoxicity of Polysaccharides. As shown in Figure 5(d), different concentrations of SLPs interact with cells for 24 h. Cell viability is greater than the normal group (100%), indicating that the polysaccharides are not toxic to cells at 20-80 μg/mL and can promote cell growth. SLP4 with the highest -OSO 3 content has the strongest effect on HK-2 cell proliferation.

Reactive Oxygen Species (ROS) Level.
A large amount of ROS is produced in damaged cells, resulting in abnormal cell function and even cell death [44]. Figure 6(a) shows the ROS fluorescence intensity of each group of cells. The intensities were detected using the DCFH-DA fluorescent probe method. The ROS fluorescence intensity in the normal group is the lowest. The ROS fluorescence intensity increases significantly after normal cells are damaged by 200 nm COD. After the addition of four kinds of polysaccharides, ROS fluorescence intensity decreases. As the -OSO 3 content of polysaccharide increases, the ROS fluorescence intensity gradually decreases. The highest degree decrease in ROS fluorescence intensity was observed after the addition of SLP4 (Figure 6(b)), owing to the protective effect of the polysaccharide. This result shows that SLPs can protect cells from damage caused by nano-COD crystals, and the protective ability of polysaccharides is positively correlated with the -OSO 3 contents of polysaccharides.
3.5.4. Cell Morphology. As shown in Figure 6(c), the cell in the normal group is evenly distributed, the cell morphology is full, and the cells are tightly connected. After the normal cells are damaged by 200 nm COD, the number of cells is significantly reduced, cell shape shrinks, and the connections between cells are destroyed. After the addition of various polysaccharides, the degree of damage caused by COD on the cells is reduced, the number of cells in the protection groups is higher than that in the damage group, the connection between cells is gradually restored, and the cell morphology is significantly improved. The ability of the polysaccharides to protect HK-2 cells from COD damage increases with -OSO 3 content of polysaccharide. The cell morphology of SLP4 with the highest -OSO 3 content is closest to the normal group.

Enhanced Antioxidant Capacities of Sulfated SLPs.
Compared with LP0, sulfated SLPs have a better scavenging ability for •OH and DPPH radicals. The reason is that the introduction of -OSO 3 groups weakens the dissociation energy of O-H bonds in polysaccharide molecules [45] and improves the hydrogen supply capacities of polysaccharide derivatives. Hydrogen atoms provided by polysaccharides combine with free radicals to form stable free radicals and terminates free radical chain reaction, thereby increasing antioxidant activity [46,47]. For example, after polysaccharides donate hydrogen atoms or single electrons to DPPH, nonradical compounds DPPH-H are generated [48]. Huang et al. [49] modified Mesona chinensis Benth polysaccharide through sulfation to obtain SMP. The scavenging rates of the two on DPPH free radicals are 75.11% and 86.95%, respectively, indicating that increase in the -OSO 3 contents of polysaccharides improve antioxidant activity. Hu et al. [50] modified Acanthopanax leucorrhizus polysaccharide (ALP) by sulfation to obtain S-ALP1 and S-ALP2 components with the substitution degrees of 0.48 and 0.73, respectively. The scavenging ability of the three on hydroxyl radicals is S-ALP2 > S-ALP1 > ALP, indicating that the antioxidant activity is positively correlated with the -OSO 3 content of polysaccharide, that is, the higher the -OSO 3 content of the polysaccharide, the stronger the antioxidant activity.

Sulfated SLPs Have a Stronger Ability to Regulate CaOx
Crystallization. Compared with the original LP0, the sulfated SLPs can better inhibit the formation of CaOx crystals (Figures 2-4), due to the following reasons.
First, the sulfated SLPs are rich in acidic -OSO 3 groups, which can better combine with the free Ca 2+ ions in the solution to form soluble complexes, increase the soluble Ca 2+ ion concentration in the system (Figure 4(b)), and reduce the amount of Ca 2+ ions combined with Ox 2-, thereby reducing the amount of CaOx precipitation, which inhibit the formation of CaOx stones [51].
Second, SLPs form polyanions in the solution and are adsorbed on the surfaces of CaOx crystals, causing defects in crystal growth, preventing free particles from entering, and inhibiting the growth of CaOx crystals. Melo et al. [52] extracted four sulfated polysaccharide components from the marine alga Dictyopteris justii. Their -OSO 3 content was 3.9%, 4.3%, 6.8%, and 7.5%, respectively. Among interfaces of the polysaccharide molecule surfaces increase. The ratio of adsorbed Ca 2+ ions leads to an increase in the energy state of free Ca 2+ ions. The high-energy interface and high-energy state Ca 2+ ions promote the formation of thermodynamic metastable COD [53]. Figure 7 shows the mechanism of SLPs inhibiting kidney stones formation. Notably, SLP3 with -OSO 3 content of 22.8% is a turning point in the mutation of a polysaccharide effect, that is, the ability of SLP3 and SLP4 to regulate the formation of COD crystals and their antioxidant activity and ability to protect cells from COD crystals are significantly greater than those of LP0, SLP1, and SLP2.
Compared with COD, COM crystals are difficult to excrete from the body because of their stronger adhesion to damaged renal epithelial cells. Therefore, sulfated SLPs that induce COD crystals are more useful in inhibiting the formation of CaOx stones than the original LP0.
SLPs with high -OSO 3 contents can inhibit the aggregation of CaOx crystals (Figure 4(a)). Aggregated crystals are not only difficult to excrete from the body but also cause considerable damage to renal epithelial cells [54,55], thereby increasing the risk of kidney stone formation. Figure 4(d) shows that the absolute value of the zeta potential of CaOx crystals generated by the regulation of sulfated SLPs is much higher than that of the original polysaccharide LP0. The surface charge density of a crystal and the amount of repulsive force between crystals increase with the absolute value of the zeta potential on the crystal surface, and the aggregation of crystals is inhibited [56].

Sulfated SLPs
Have a Strong Ability to Protect HK-2 Cells from Crystal Damage. The biological activity of sulfated polysaccharides is closely related to their -OSO 3 content.
Compared with LP0, sulfated SLPs can better protect HK-2 cells from the damage of nano-COD crystals. The results of this paper show that the ability of SLPs to protect cells from crystals damage is positively correlated with their -OSO 3 content ( Figure 5(e)). As the -OSO 3 content of SLPs increases, cell viability gradually increases ( Figure 5(c)), ROS levels decrease (Figure 6(a)), and cell morphology gradually recovers (Figure 6(c)). The ability of SLP3 and SLP4 to protect cells from COD crystal damage is significantly greater than that of LP0, SLP1, and SLP2.
Jin et al. [57] found that sulfation modification can improve the antioxidant activity of polysaccharides and can better protect cells from oxidative damage and apoptosis induced by H 2 O 2 . Wang et al. [58] showed that compared with the original Cyclocarya paliurus polysaccharides, sulfated polysaccharides have a better protective effect on oxidative stress caused by H 2 O 2 . Moreover, Wang et al. [59] proved that sulfation modification can enhance the immune activity of Lycium barbarum polysaccharide.

Conclusions
In this study, the original LP0 was sulfated using the sulfur trioxide-pyridine method, and four kinds of sulfated polysaccharide SLPs with the -OSO 3 − contents of 8.58%, 15.1%, 22.8%, and 31.3% were obtained. As the -OSO 3 content of polysaccharides increases, its regulating effect on the growth of CaOx crystals is enhanced. As a result, the concentration of soluble Ca 2+ in the solution increases, the amount of CaOx crystal precipitation is reduced, the degree of CaOx crystal aggregation is significantly reduced, and the percentage of induced COD crystals increases. These effects can effectively inhibit the formation of CaOx kidney stones.