Ang II was purchased from Abcam (Cambridge, United Kingdom). Isoflurane was obtained from Ruiwode Life Technology Co., Ltd. (Shenzhen, Guangdong, China). Hematoxylin and eosin were purchased from Solarbio Technology Co., Ltd., (Beijing, China). terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) Apoptosis Detection Kit I POD was purchased from Boster Biological Technology Co., Ltd. (Wuhan, Hubei, China). Annexin V-AbFluor™ 647 Apoptosis Detection Kit (cat. no. KTA0004), Cell Counting Kit-8 (CCK8), and antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cat. no. Abp57259) were obtained from Abbkine (Wuhan, Hubei, China). Radioimmunoprecipitation assay (RIPA) lysis buffer, primary antibody dilution buffer, and secondary antibody dilution buffer were supplied by Beyotime Biotechnology (Shanghai, China). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore (Billerica, MA, United States). Antibodies against Bcl-2 (cat. no. 12789-1-AP) and p53 (cat. no. 10442-1-AP) were purchased from ProteinTech (Rosemont, IL, United States). Antibodies against BAX (cat. no. 2772), caspase-3 (cat. no. 9662), and caspase-9 (cat. no. 9508) and secondary antibodies anti-mouse (cat. no. 7076) and anti-rabbit (cat. no. 7074) were purchased from Cell Signaling Technology (Danvers, MA, United States). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA, and bicinchoninic acid (BCA) protein assay reagent kit were supplied by Thermo Fisher Scientific (Waltham, MA, United States).

QDG is composed of four herbs: G. elata Blume (Tianma), U. rhynchophylla (Miz.) Miz. ex Havil. (Gouteng), S. baicalensis Georgi (Huang Qin), and N. nucifera Gaertn (Lianzixin) in a ratio of 12:10:6:5. QDG was extracted and supplied by Tianjiang Pharmaceutical Co., Ltd. (Jiangsu, China).

For the animal experiment, QDG was prepared as in our previous studies. The concentrations of QDG were selected based on our previous study (Cheng et al., 2021). Briefly, QDG power was dissolved in double-distilled water (dd H₂O) to the indicated concentration immediately before use. For cell culture experiments, the stocking solution of QDG was prepared in serum-free medium to 100 mg/ml and was prepared with completed medium to indicated concentrations of QDG before use.

Male C57BL/6 mice (28 ± 1 g, 8 weeks) were purchased from SLAC Laboratory Animal Technology Co., Ltd. (Shanghai, China). All mice were raised under specific pathogen-free conditions, with controlled humidity (50%–60%) and temperature (23 ± 1°C), 12 h of light/dark cycle, and free access to food and water. After 1 week of adaptation, the mice were randomly divided into three groups (n = 5 each): Control, Ang II, and Ang II + QDG groups. Mice in the Ang II or Ang II + QDG group were infused with Ang II (500 ng/kg/min), while mice in the Control group were infused with saline for 4 weeks by subcutaneously implanting micro osmotic pumps. Mice in the Ang II + QDG group were intragastrically administrated with QDG daily (1.145 g/kg/day; 200 μl for each mouse), whereas mice in both Control and Ang II groups were intragastrically administrated with equal volumes of dd H₂O for 4 weeks. The animal experimental protocols of this study were approved by the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine and were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

The blood pressure, including systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), was measured prior to the experiment and once a week for 4 weeks by using the tail-cuff plethysmograph method with the CODA™ noninvasive blood pressure system (Kent Scientific, Torrington, CT, United States).

Hematoxylin–eosin (HE) staining was performed to evaluate the morphological changes of renal tissues from each group. Briefly, renal tissues were fixed with 4% paraformaldehyde solution for 48 h, then embedded in paraffin, cut into 4 µm sections, and stained with hematoxylin for 1 min and eosin for 2 s. Then, the sections were visualized under an optical microscope (Leica, Wetzlar, Germany) at a magnification of ×400.

The renal tissues were stored in RNA later (Takara, Beijing, China) at room temperature for 1 h and moved to −20°C for long-time storage. Total RNA was extracted by a TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. The concentration and quality of total RNA were measured by both Qubit 3.0 and Agilent 2100 Bioanalyzer. RNA samples with a RIN value of 7 or higher were used for further experiments.

RNA-seq was performed at Capital Bio Technology (Beijing, China). In brief, rRNA was removed from total RNA by a NEB Next rRNA Depletion Kit (NEB, Ipswich, MA, United States) according to the instructions. rRNA-depleted total RNA was fragmented, and the poly(A)-tailed mRNA molecules was generated by a NEB Next Ploy(A) mRNA Magnetic Isolation Module Kit (NEB, Ipswich, MA, United States) according to the manufacturer’s instructions. Moreover, the final libraries were quantified on an Agilent 2100 Bioanalyzer using a KAPA Library Quantification Kit (KAPA Biosystem, Wilmington, MA, United States) and subjected to paired-end sequencing on an Illumina HiSeq sequencer (Illumina, San Diego, CA, United States).

The raw data were processed as described previously (Wu et al., 2021). The selected genes were further analyzed in the context of the information obtained from the database of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).

The renal tubular epithelial cell line NRK-52E was purchased from BeNa (Beijing, China) and cultured in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. The cell lines were cultured in a humid environment at 37°C and 5% CO₂. The NRK-52E cells were cultured in six-well plates at a density of 0.8 × 10⁵ cells per well. After being cultured for 24 h, NRK-52E cells were incubated in minimum medium (0.5% FBS) for around 8 h and were divided into three groups: Control group, Ang II group, and Ang II + QDG group. Then, cells in Ang II and Ang II + QDG groups were incubated with Ang II (1 μM), followed by treatment with QDG (25 and 50 μg/ml) in the Ang II + QDG group for 48 h.

To assess cell apoptosis in renal tissues from each group, renal tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm, and stained using a TUNEL kit according to the manufacturer’s instructions. The specific experimental procedures for TUNEL assay were performed as previously described (Cheng et al., 2021). Slides were visualized under an optical microscope (Leica, Wetzlar, Germany) at a magnification of ×400, and five fields of view were randomly selected for each slide. Positively stained cells in each field were determined using ImageJ software.

For NRK-52E cells with or without Ang II stimulation and QDG treatment, the cells were fixed in 4% paraformaldehyde and incubated for 30 min at room temperature. The next step was the same as described above. Cells with a dark-brown nucleus were defined as apoptotic cells, while cells without or with a light-brown nucleus were defined as normal (non-apoptotic cells). The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells examined.

The cell viability of NRK-52E cells was determined by CCK8. Briefly, the cells were seeded into 96-well plates (2 × 10³ per well) containing complete culture medium and cultured at 37°C in 5% CO₂ overnight. The indicated concentrations of QDG were added to each well for 48 h. At the end of treatment, a CCK8 reagent (10 µl per well) was added to each well and incubated at 37°C for 2 h in the dark. The absorbance was determined by an enzyme-labeling instrument (Multiskan FC, Thermo Fisher Scientific) at 450 nm wavelength. The cell viability of untreated cells was set as 100%.

To provide a comparative assay of apoptosis by Annexin V staining, the NRK-52E cells were seeded into six-well plates at a density of 0.8 × 10⁵ cells per well. After being cultured for 24 h, NRK-52E cells were cultured in serum-free DMEM for 12 h and then were stimulated with Ang II (1 μM) and treated with or without QDG (25 μg/ml or 50 μg/ml) in 0.5% of minimum medium (0.5% FBS) for a total of 24 h. At the end of treatment, cells were harvested and stained with Annexin V and propidium iodide (PI) according to the manufacturer’s protocol. Cell apoptosis was detected and quantified by FACS (fluorescence-activated cell sorting) analysis.

The cell lysis buffer with protease inhibitor and PMSF was added to the renal tissues, which were then ground with a low-temperature grinder, applied on ice for 30 min, and centrifuged at 4°C and 14,000 g for 15 min. The protein concentration was quantified by BCA assay. A total of 50 μg protein from each sample was separated on 10% SDS-PAGE gel, transferred to PVDF membranes, and blocked with 0.5% bovine serum albumin for 2 h followed by incubation with the primary antibodies (overnight at 4°C), including rabbit anti-BAX (1:1,000), rabbit anti-BCL2 (1:1,000), rabbit anti-p53 (1:1,000), rabbit anti-cle-caspase-3 (1:1,000), rabbit anti-GAPDH (1:5,000), and mouse anti-cle-caspase-9 (1:1,000). Then, the membrane was washed with TBST and then incubated with secondary antibody at room temperature for 1 h. At the end of incubation, the membranes were washed with TBST, and then detection was performed using an electrochemiluminescence (ECL) kit. The expression of GAPDH protein was used as an internal control. The protein levels were analyzed with ImageJ software.

The data were presented as mean ± SD. All statistical analyses were performed using the SPSS software (Version 22.0) for Windows (SPSS, Inc., Chicago, IL, United States). One-way analysis of variance was used to compare differences among three groups, when conforming to a normal distribution. The nonparametric Kruskal–Wallis test was used to compare differences among the three groups when not consistent with the normal distribution. A p-value <0.05 was considered as statistically significant.

The effect of QDG on hypertension-induced renal injury was assessed using Ang II-infused mice. Consistent with our previous studies (Na et al.; Zhang et al., 2020; Cheng et al., 2021; Wu et al., 2021), Ang II infusion significantly increased the blood pressure, including SBP (Figure 1A; *p < 0.05, vs. the Control group), DBP (Figure 1B; *p < 0.05, vs. the Control group), and MAP (Figure 1C; *p < 0.05, vs. the Control group) of mice, which were attenuated after QDG treatment. Moreover, HE staining revealed significant renal pathological changes, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilatation, which were alleviated after QDG treatment (Figure 1D). These results suggest that QDG treatment might relieve Ang II-induced hypertension and renal injury in Ang II-infused mice.

To explore the underlying mechanisms of QDG on hypertensive renal protection, RNA-seq was performed to identify the differentially expressed transcripts (DETs) in renal tissues among different groups (GSE182517; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182517). As shown in Figures 2A,B (left panels), Ang II infusion significantly upregulated the expression of 1,427 transcripts and downregulated the expression of 1,173 transcripts in renal tissues of mice, compared to the Control group, while QDG treatment significantly upregulated the expression of 868 transcripts and downregulated the expression of 868 transcripts, compared with the Ang II group (Figures 2A,B, right panel). The integrative analysis between the two comparisons (Ang II vs. Control and Ang II + QDG vs. Ang II) identified 261 downregulated transcripts (Figure 2C, left panel) and 401 upregulated transcripts (Figure 2D, right panel) in the Ang II group, which were reversed after QDG treatment.

GO enrichment analysis based on the DETs between Ang II and Control groups revealed multiple predicted potential functions. As shown in Figure 3A, cellular component (CC) analysis indicated that DETs were mainly involved in the intracellular part, intracellular membrane bounded organelle, and mitochondrion. Biological process (BP) analysis showed that DETs were mainly associated with metabolism, gene expression, and organelle organization. In the molecular function (MF) category, DETs were significantly enriched in binding, translation factor activity, etc. Moreover, GO enrichment analysis based on the DETs between Ang II + QDG and Ang II groups revealed multiple predicted potential functions. As shown in Figure 3B, CC analysis showed that DETs were associated with the cytoplasmic part, intracellular part, and cell part. As to BP, DETs were significantly enriched in the catabolic process, amide biosynthesis, and translation. The MF analysis for these DETs includes binding, catalytic activity, and translation elongation factor activity.

KEGG pathway analysis based on the DETs between Ang II and Control groups revealed multiple enriched signaling pathways, including apoptosis, p53 signaling pathway, and MAPK signaling pathway (Figure 4A). Moreover, KEGG pathway analysis based on the DETs between Ang II + QDG and Ang II groups demonstrated multiple enriched signaling pathways, including oxidative phosphorylation, apoptosis, and p53 signaling pathways (Figure 4A). The integrative analysis between the two comparisons (Ang II vs. Control and Ang II + QDG vs. Ang II) demonstrated that multiple signaling pathways were enriched in both comparisons (Figure 4B). Notably, we find that both cell apoptosis and p53 signaling pathways were significantly enriched in both comparisons (Figure 4C), which encouraged us to further explore the regulatory effects of QDG on cell apoptosis and activation of the p53 signaling pathway in renal tissues of Ang II-infused mice.

Further verification of QDG treatment on cell apoptosis of renal tissues by performing TUNEL staining revealed an increase of the percentage positively TUNEL-stained cells in renal tissues of Ang II-infused mice (Figures 5A,B; *p < 0.05 vs. the Control group), while QDG treatment significantly reduced the percentage of positively TUNEL-stained cells (Figures 5A,B; #p < 0.05, vs. the Ang II group), confirming the effect of QDG on attenuation cell apoptosis in renal tissues in Ang II-infused mice. We further assessed the effect of QDG treatment on cell apoptosis of a rat renal tubular epithelial cell line (NRK-52E) by performing TUNEL assay and found that 25 and 50 μg/ml of QDG treatment did not affect the cell viability of NRK-52E (Figure 5C) but attenuated the Ang II stimulation-induced cell apoptosis of NRK-52E cells (Figures 5D,E; *p < 0.05, vs. the Control group, #p < 0.05, vs. the Ang II group). Consistently, by performing Annexin V/PI staining, we found that Ang II stimulation significantly increased the percentage of cell apoptosis, which was attenuated after QDG treatment (Figures 5F,G; *p < 0.05, vs. the Control group, #p < 0.05, vs. the Ang II group).

To further verify the regulatory effect of QDG treatment on the activation of the p53 pathway, western blot confirmed that Ang II infusion significantly upregulated the protein expression of p53, which was downregulated after QDG treatment. Moreover, by determination of the expression apoptosis-related proteins using western blot analysis, it was revealed that Ang II infusion significantly upregulated the expression of proapoptotic proteins Bax, cle-caspase-9 and cle-caspase-3 but downregulated the expression of antiapoptotic protein Bcl-2 (Figure 6; *p < 0.05, vs. the Control group), which were all reversed after QDG treatment (Figure 6; #p < 0.05, vs. the Ang II group).