Four New Benzoylamide Derivatives Isolated from the Seeds of Lepidium apetalum Willd. and Ameliorated LPS-Induced NRK52e Cells via Nrf2/Keap1 Pathway

Four new benzoylamide derivatives, lepidiumamide B–E (1–4), were isolated from the seeds of Lepidium apetalum Willd. The structures were determined by a combination of MS and NMR analyses. All compounds were evaluated for their protective effects against NRK-52e cell injury induced by lipopolysaccharide (LPS) in vitro. These compounds showed significantly protective activity and ameliorated LPS-induced NRK52e cells via the Nrf2/Keap1 pathway. The discovery of these active compounds is important for the prevention and treatment of renalinjury.


Effects of Compounds 1-4 on LPS-Induced NRK-52e Cell Viability
As shown in Figure 3, the NRK-52e cell viability was reduced by LPS, and compounds 1-4 could effectively enhance the cell viability of NRK-52e induced by LPS.

Effects of Compounds 1-4 on LPS-Induced NRK-52e Cell Viability
As shown in Figure 3, the NRK-52e cell viability was reduced by LPS, and compounds 1-4 could effectively enhance the cell viability of NRK-52e induced by LPS.

Effects of Compounds 1-4 on the Levels of Keap1 and p-Nrf2 in NRK-52e Cell Induced by LPS
As shown in Figure 4, the level of Keap1 was increased and the level of p-Nrf2 was decreased in LPS-induced NRK-52e cells, which could be reversed by compounds 1-4.

Effects of Compounds 1-4 on the Levels of Keap1 and p-Nrf2 in NRK-52e Cell Induced by LPS
As shown in Figure 4, the level of Keap1 was increased and the level of p-Nrf2 was decreased in LPS-induced NRK-52e cells, which could be reversed by compounds 1-4.

General Experimental Procedures
NMR spectra were recorded at room temperature on a Bruker Avance III 500 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany). Optical rotations were performed on an APIV (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectroscopy was determined on a Nicolet iS10 Microscope Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). HRESIMS spectra were obtained on a Bruker maXis HD mass spectrometer. UV spectra were obtained on a Shimadzu UV-2401PC apparatus (Shimadzu, Kyoto, Japan

Cell Culture and MTT Assay
The NRK-52e cell line was purchased from ATCC (MD, USA) and maintained in DMEM supplemented with 10% FBS and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) in a humidified atmosphere of 5% CO 2 at 37 • C.

Immunofluorescence
The NRK-52e cells were fixed with 4% paraformaldehyde for 15 min and washed with ice-cold PBS. Then, they were incubated with 0.25% Triton X-100 in PBS for 10 min and rinsed three times with PBS for 5 min. For immunofluorescence staining, the cells were blocked in PBST with 2% bovine serum albumin (BSA, Genview; Beijing, China) for 30 min, followed by incubation with primary antibodies Keap1 (ab139729, Abcam, MA, USA) and p-Nrf2 (ab76026, Abcam) overnight at 4 • C. After washing with PBS, the cells were stained with Alexa Fluor TM 594 donkey anti-rabbit IgG (H + L) (1981155, Thermofisher) for 1 h in the dark and rinsed 3 times with PBS for 5 min. The cells were then incubated with DAPI for 3 min and then rinsed with PBS. Finally, the plate was scanned using Cytation 5 (BioTek, Shoreline, WA, USA) [23].

Statistical Analysis
Data were analyzed using SPSS 26.0 software (IBM; Endicott, NY, USA). Statistical significance was assessed in comparison with the respective control for each experiment using one-way analysis of variance. A p value less than 0.05 indicated a statistically significant difference.

Conclusions
In this study, four new benzoylamide derivatives, lepidiumamide B-E (1-4) were isolated from the seeds of L. apetalum. The structures were determined by a combination of MS and NMR analyses. The isolated compounds were evaluated for their protective effects against NRK-52e cell injury induced by LPS in vitro and showed significantly protective activity, as well asameliorated LPS-induced NRK52e cells via the Nrf2/Keap1 pathway.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the manuscript.