Scavenging ROS to Alleviate Acute Liver Injury by ZnO‐NiO@COOH

Abstract Currently, the incidence of acute liver injury (ALI) is increasing year by year, and infection with coronavirus disease 2019 (COVID‐19) can also induce ALI, but there are still no targeted therapeutic drugs. ZnO–NiO particles is mainly used to clean up reactive oxygen species (ROS) in industrial wastewater, and it is insoluble in water. Its excellent properties are discovered and improved by adding shuttle‐based bonds to make it more water‐soluble. ZnO‐NiO@COOH particles are synthetically applied to treat ALI. The p‐n junction in ZnO–NiO@COOH increases the surface area and active sites, thereby creating large numbers of oxygen vacancies, which can quickly adsorb ROS. The content in tissues and serum levels of L‐glutathione (GSH) and the GSH/oxidized GSH ratio are measured to assess the capacity of ZnO–NiO@COOH particles to absorb ROS. The ZnO–NiO@COOH particles significantly reduce the expression levels of inflammatory factors (i.e., IL‐1, IL‐6, and TNF‐α), macrophage infiltration, and granulocyte activation. ZnO‐NiO@COOH rapidly adsorb ROS in a short period of time to block the generation of inflammatory storms and gain time for the follow‐up treatment of ALI, which has important clinical significance.


Supplemental Materials and Methods 2
Hematoxylin & Eosin (HE) staining Tissues slices were dewaxed by heating to 62°C in an oven for 2 h and then rehydrated in a graded series of xylene/alcohol/PBS (xylene Ⅰ, 10 min → xylene Ⅱ, 10 min → 100% alcohol Ⅰ, 5 min → 100% alcohol II, 5 min → 95% alcohol I, 5 min → 95% alcohol II, 5 min → 85% alcohol, 5 min → 75% alcohol, 5 min → PBS I, 5 min). Afterward, the tissues slices were stained with hematoxylin for 10 min. Color separation was performed by rinsing the tissue slices with 1% hydrochloric acid and alcohol (300 mL of 75% alcohol, 2 mL of concentrated HCl) for 30 s followed by running tap water for 5 min. Next, the tissues were stained with eosin dye solution for about 50 s and then rinsed with 95% alcohol. The slices were then subjected to a graded series of alcohol and xylene for dehydration and transparency (75% alcohol, 5 min → 85% alcohol, 5 min → 95% alcohol, 3 min → 100% alcohol, 3 min → xylene I, 10 min → xylene II, 10 min). Afterward, the slices were mounted onto glass slides with neutral gum and observed under a microscope.

TUNEL analysis
The tissue slices were dewaxed by heating to 62°C in an oven for 2 h and then rehydrated in a graded series of xylene/alcohol/PBS (xylene I, 10 min → xylene II, 10 min→ 100% alcohol I, 5 min→ 100% alcohol II, min → 95% alcohol I, 5 min → 95% alcohol II, 5 min → 85% alcohol, 5 min → 75% alcohol, 5 min → PBS I, 5 min → PBS II, 5 min). The slides were immersed in 3% H 2 O 2 to block endogenous peroxidase activity, incubated for 20 min at room temperature, and then washed three times with PBS for 5 min each time. Following the addition of proteinase K at 37°C for 30 min, the slides were washed twice with PBS for 5 min each time.
The TUNEL reaction mixture consisted of 50 μL of TdT enzyme and 450 μL of dUTP.
Experimental group: 100 μL of the TUNEL reaction mixture.
Positive control group: 100 μL DNase I solution at room temperature for 10 min, then 100 μL of the TUNEL reaction mixture.
Negative control group: 50 μL of dUTP solution.
The sections were incubated at 37°C for 1 h in the dark. At the end of the reaction, the samples were washed three times with PBS for 5 min each time. Next, 50 μL of converter-POD working solution were added and the samples were incubated at 37°C in the dark for 60 min, then washed with PBS three times for 5 min each time. Color was developed by dropwise addition of 50 μL of DAB color developing solution and reacted for 10 min at room temperature, then washed three times with PBS for 5 min each time. The nuclei were counterstained with hematoxylin. Finally, the samples were mounted on glass slides with neutral gum and observed under a microscope.

Supplemental Materials and Methods 4
The mRNA extraction Before the experiment, all relevant instruments were treated with DEPC water to remove RNase and then autoclaved. The tissue fragments were placed in 1.5 mL Eppendorf tube and mixed with 1 mL of Trizol reagent, then placed on ice for 5 min to fully lyse the cells. Afterward, the tissues were disrupted by shaking with magnetic beads at 300 rpm for 5 min. Following centrifugation at 12,000 rpm at 4°C for 5 min, the supernatant was transferred to a new Eppendorf tube, then mixed with 200 μL of chloroform, shaken vigorously for 15 s, and placed on ice for 10 min. Afterward, the samples were centrifuged at 12,000 rpm and 4°C for 15 min. The homogenate in the tube was divided into three layers with the RNA dissolved in the colorless upper layer, which was transfer to a new eppendorf tube. Following the addition of an equal volume of isopropanol, the sample was mixed well and then incubated at room temperature for 10 min. Following centrifugation at 12,000 rpm at 4°C for 10 min, the supernatant was removed. Then, the RNA pellet was washed with 1 mL of 75% ethanol and centrifuged at 12,000 rpm and 4°C for 5 min. After the ethanol was removed, the tube was incubated at 55°C for 10 min. Next, the RNA was suspended in 20-50 μL of RNase-free DEPC water. The concentration of the RNA was measured at 260 nm and purity at 280 nm, expressed as A260/A280. Finally, the samples were stored at -80°C until use.

Supplemental Materials and Methods 5
Reverse Transcription RNA (2 μg), N6 primer (1 μL), and DEPC water to a final volume of 15 μL were mixed well in a 70°C water bath for 5 min, and then cooled on ice for 2 min.

Supplemental Materials and Methods 6
Real-time fluorescent quantitative PCR The Gene IDs of the target genes of the required species were retrieved from the GeneBank database and the primers were obtained from PrimerBank (Supplemental Table 1). Each 10 μL reaction system consisted of SYBR Green I (dye (5 μL), forward primer (0.5 μL), reverse primer (0.5 μL), cDNA (1 μL), and DEPC water (3 μL). After the above reaction system was fully mixed and centrifuged, the PCR reaction was carried out according to the following reaction conditions: pre-denaturation at 95°C for 30 s followed by 40 cycles of amplification at 95°C for 5 s and 60°C for 32 s.
Dissolution curve program: 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s for 1 cycle.
The purity of the amplified products was determined by reference to the melting curve.
According to dynamic analysis, the relative expression of target gene mRNA was calculated with the following formula: 2 -∆∆Ct =2 -∆Ct Object group-∆Ct control group

Immunohistochemical analysis
The slices were dewaxed by heating to 60°C in an oven for 2 h and then deparaffinized with a graded series of xylene/ethanol/water (xylene, 10 min → xylene, 10 min → xylene, 10 min → absolute ethanol, 10 min → 95% ethanol, 5 min → 85% ethanol, 5 min → 75% ethanol, 5 min → ddH 2 O, 5 min). Endogenous peroxidase was inactivated by immersing the slices in 3% H 2 0 2 for 30 min. Antigen retrieval was performed with acid retrieval solution (21.9 g of trisodium citrate, 3.6 g of citric acid, and ddH 2 O to a final volume of 3,000 mL). Next, blocking was performed with 1% BSA at 37°C for 30 min. Then, the slices were incubated overnight with primary antibodies (dilution, 1:500-10,000) at 4°C followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 37°C for 30 min. Color was developed with the use of DAB solution for 3-10 min. The nuclei were stained with hematoxylin for 10 min (differentiation 1-2 times). The slices were dehydrated with a graded series of ethanol/xylene/phenolic acid (75% ethanol, 5 min → 85% ethanol, 5 min → 95% ethanol, 10 min → absolute ethanol, 10 min → phenolic acid, 5 min → xylene, 10 min → xylene, 10 min). Afterward, the slices were sealed in resin and stored for later use.