Animals and drugs: 72 SPF balb/c male mice, aged at 4–6 weeks, weighed at 18–20 g (Experimental Animal Center of China Three Gorges University, Yichang, China). HBG (Cat# 200568, Xinjiang Uygur Pharmaceutical Co., Ltd., Wulumuqi, China). Cisplatin (Cat# N0624A, Dalian Meilun Biotechnology Co., Ltd., Dalian, China). SKP (Cat# JP17095, Tianjin Tongrentang Group Co., Ltd., Tianjin, China). The standards of quercetin (Cat# 19082203), kaempferol (Cat# 20082105), quercitrin (Cat# 18082102), apigenin (Cat# PS011063) and chlorogenic acid (Cat# 516E023) were acquired from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China).

Kits and instruments: Cr (Cat# C011-2-1) and BUN (Cat# C013-2-1) kit (Nanjing Jiancheng Pharmaceuticals, Nanjing, China). MDA (Cat# A003-1-2), GSH (Cat# A005-1-2), SOD (Cat# A001-3-2) and CAT (Cat# A007-1-1) kits (Nanjing Jiancheng Pharmaceuticals, Nanjing, China). Mouse TNF-$\alpha$, IL-6, IL-1$\beta$ ELISA kit (Cat# E-EL-M3063, Cat# E-EL-M2453c, Cat# E-EL-H0149c, Elabscience, China). ECL luminescent liquid (Cat# MA0186-1, Dalian Meilun Biotechnology Co., Ltd., Dalian, China). An Agilent 1260 Infinity system (Agilent Technologies, USA) was used for the High Performance Liquid Chromatography (HPLC) analysis. Enzyme-labelled instrument (SPARK 10M, TECAN, Switzerland). High speed centrifuge (Centrifuge 5810R, Eppendorf, Germany). Chemiluminescence gel imaging system (NIKON DS-U3, NIOKN, Japan).

Accurately weighing 1.6 g of HBG was dissolved in 80% methanol, extracted by ultrasonic wave (power: 350 W, frequency: 53 kHz) for 40 min, and fixed the volume to 10 mL. The organic microporous membrane was used for filtration, and the filtrate was the test solution.

The HPLC of Agilent 1260 was used, Eclipse XDB-C${}_{18}$ (4.6 mm $\times$ 250 mm, 5 µm), setting the elution solvent A as acetonitrile, B as 0.1% formic acid water, the gradient elution regimen was designed as follows: 0$\backsim$15 min, A for 5% $\to$ 10%, 15$\backsim$35 min, A for 10% $\to$ 18%, 35$\backsim$50 min, A for 18% $\to$ 25%, 50$\backsim$60 min, A for 25% $\to$ 35%, 60$\backsim$70 min, A for 35% $\to$ 70%. The UV was 327 nm.

72 Balb/c mice were caged in a climate-controlled room under specified SPF conditions. Day and night were alternated for 12 h. This research project was approved by the Ethics Committee of Hubei University of Traditional Chinese Medicine (NO. HUCMS202006011). The mice were randomly divided into six groups before the experiment. The control group received oral saline for eleven consecutive days, and a single intraperitoneal (i.p.) injection of saline was given on the seventh day. The cisplatin group received oral saline for eleven consecutive days, and a single i.p. injection of cisplatin (13 mg/kg) was also given on the seventh day. HBG groups: For eleven days straight, HBG (1.6, 3.2, or 6.4 g/kg/day) was given orally once, and on the seventh day, cisplatin was applied intraperitoneally just once. SKP group: For eleven days straight, SKP (1.2 g/kg/day) was administered orally. On the seventh day, cisplatin was delivered intraperitoneally once. The final day, all mice were gavaged, sedated with 0.3% sodium pentobarbital (80 mg/kg body weight), and then put to death. All subsequent test samples were 10. The remaining kidneys were stored in reserve at –80 °C while a portion of the kidneys were removed and fixed in 4% paraformaldehyde.

The weight of the mice in each group was recorded daily and all behavioral traits were scrutinized. The organ coefficient was computed after the liver and kidney of mice were weighed.

The blood sample was centrifuged. Upper serum was taken out and kept at –80 °C. A commercial kit was utilized to detect the contents of serum Cr and BUN in accordance with the manufacturer’s instructions.

The kidneys were taken out of 4% PFA, encased in paraffin, dehydrated with ethanol, and removed with xylene. H&E staining was applied to paraffin sections (5 µm) before being inspected under a light microscope (NIKON ECLIPSE CI, Tokyo, Japan).

The frozen sections of kidney were incubated in dark with ROS staining solution. The nuclei were stained with DAPI. A quenching agent for anti-fluorescence was employed to seal the film. Through using fluorescent microscope, the images were inspected and recorded.

The kidney and ice-cold normal saline were homogenized and centrifuged (1:10, w/v). Using commercial kits in accordance with the directions, the resulting supernatant was utilized to assess the concentrations of MDA, reduced GSH, SOD, and CAT in conjunction to their contents and activities.

The kidney tissue’s apoptosis was observed by using the TUNEL assay. After already being treated with proteinase K, kidney slices were cultured in a solution of nucleotides that contained both fluorescein dUTP and TDT. DAPI was applied to stain the nuclei. A quenching agent for anti-fluorescence was added to seal the film. Through using fluorescent microscope, the images were inspected and recorded.

TNF-$\alpha$, IL-1$\beta$, and IL-6 content in serum were calculated by utilizing ELISA kit.

After rehydrating and eliminating the paraffin from the kidney slices, endogenous peroxidase was blocked before being coated with BSA. A specific amount of the primary antibody made with PBS was dripped on the sections, which were then incubated overnight at 4 °C. PBS was utilized to rinse the sections. The sections were then covered with a second antibody from the relevant species, which was HRP-labeled, and they were allowed to legally sit at room temperature for 50 minutes. PBS was employed to rinse the sections. To parts, DAB chromogenic solution was used to produce color. Harris hematoxylin was dyed for about 3 min and used tap water to cleanse for 10 min. After being dried and sealed with neutral gum, the pieces were sanitized with ethanol and xylene. Scrutiny under a microscope, image gathering, and evaluation.

When creating a protein sample from kidney tissue, 5% 2-ME activation buffer and 5% loading buffer were added. The protein samples were placed onto an electrophoresis gel made of 10% SDS-polyacrylamide. The proteins were then transferred at 300 mA for 60 min to polyvinylidene difluoride membranes. The membranes were submerged in 5% skim milk (Sigma) for two hours. The membrane was in interaction with the diluted primary IL-18, IL-1$\beta$, IL-6, and TNF-$\alpha$ antibodies for a number of hours. The membrane was detected for an hour with the suitable secondary antibody after being washed in TBST. The polyvinylidene difluoride membranes were colored by ECL luminescent liquid and was developed and photographed by chemiluminescence gel imaging system.

One-way ANOVA and post hoc examination were utilized to contrast the results to the cisplatin group. Estimated to be a noteworthy value was p 0.05. GraphPad Prism 8.0 (GraphPad Software, USA) was implemented to conduct the statistic evaluations.

As shown in Fig. 1, HPLC of HBG and reference standard (327 nm), 1 is Chlorogenic acid, 2 is Quercetin, 3 is Kaempferol, 4 is Apigenin, 5 is Quercitrin.

Fig. 1.

High Performance Liquid Chromatography (HPLC) of Hugan Buzure Granule (HBG) and reference standard (327 nm). 1 is Chlorogenic acid, 2 is Quercetin, 3 is Kaempferol, 4 is Apigenin, 5 is Quercitrin.

The mice of Cisplatin group were dispirited, their hair color was dim and lusterless, their weight dropped sharply, their drinking water and eating decreased, and some mice had loose stools and their stomachs were swollen (Fig. 2A). The mental state of the mice in the HBG groups was noticeably better than that in the Cisplatin groups, their hair was smoother and their diet and drinking water were normal compared to the Cisplatin groups, and the phenomena of loose stools was diminished. The recovery of mice in Cisplatin + 6.4 g/kg HBG group was the fastest, and that in SKP group was the same as that in Cisplatin + 3.2 g/kg HBG group. The kidneys in the Control group were slender and long, and they were a dark red color. The kidneys in the Cisplatin group, in contrast hand, were plainly enlarged, and they were a lighter red color than the kidneys in the Control group (Fig. 2B). In HBG group, the enlargement of kidney was alleviated and the color gradually became dark. In contrast to the Control group (Fig. 2C), the Cisplatin group’s kidney coefficient increased considerably (p 0.01); The renal coefficient of HBG groups was markedly smaller than that of the Cisplatin group (p 0.01). Relatively, the liver coefficient of Cisplatin group and HBG groups did not change significantly (Fig. 2D).

Fig. 2.

The effect of HBG on organ coefficient and serum Creatinine (Cr) and Blood Urea Nitrogen (BUN) in Chronic Kidney Injury (CKI) mice. (A) The weight of mice changed with time. (B) Appearance of kidney in AKI mice. (C) Kidney coefficient. (D) Liver coefficient. (E) Serum Cr. (F) Serum BUN. Data were expressed as the mean $\pm$ SD. ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. the control group. **p 0.01 vs. the Cisplatin group.

As shown in Fig. 2E,F, the serum Cr and BUN were considerably higher in Cisplatin group (p 0.01). In comparison to the Cisplatin group, the serum Cr and BUN levels were considerably lower in the HBG and SKP groups (p 0.01).

The morphology of glomeruli and renal tubules was healthy, the structure of kidney tissue cells was ordinary, the arrangement of renal tubular cells was ordered and tight, and the boundary between cells was visible (Fig. 3A). The structure of kidney tissue in the Cisplatin group was obviously destroyed, many large vacuoles could be seen, the renal tubules were seriously dilated, and the arrangement of renal tubular cells was scattered. In comparison to Cisplatin group, the histological characteristics of the kidney injury in HBG groups were significantly improved, the number of vacuoles decreased, the expansion of renal tubules decreased, and the arrangement of renal tubular cells tended to be orderly. The Cisplatin + 6.4 g/kg HBG group showed more significant injury reduction. The recovery of SKP group was similar to that of Cisplatin + 3.2 g/kg HBG group.

Fig. 3.

HBG influences the morphological change of kidney tissues in CKI mice. (A) H&E staining section of kidney tissues ($\times$200). (B) TUNEL assay of kidney tissues ($\times$200). (C) ROS staining of kidney tissues ($\times$200).

As shown in Fig. 3B, brown was positive expression, indicating apoptosis. The positive expression of Cisplatin group was considerably increased. The positive expression of HBG groups was noticeably reduced, while the positive expression of SKP groups was even more noticeably reduced.

In contrast to the Control group, as seen in Fig. 3C, the fluorescence intensity of the Cisplatin group was much greater, indicating that the amount of ROS was dramatically higher. Compared with Cisplatin group, the fluorescence intensity was noticeably reduced in HBG groups, and the most obvious decrease was found in Cisplatin + 6.4 g/kg HBG group, the amount of ROS in SKP group was as similar to that in cisplatin + 3.2 g/kg HBG group.

As seen in Fig. 4A–D, the Cisplatin group’s MDA content increased distinctly (p 0.01), while the GSH content diminished markedly (p 0.05). MDA levels in the HBG and SKP groups considerably dropped (p 0.01), whereas GSH contents climbed. The activities of CAT and SOD in the Cisplatin group were considerably reduced (p 0.01). The CAT and SOD activities in the HBG and SKP groups swelled notably (p 0.01).

Fig. 4.

The level of oxidative stress associated to indicators in AKI mice’ kidney tissue. (A) is the MDA content, (B) is the GSH level, (C) is the activity of CAT, (D) is the content of SOD, (E) is the serum TNF-$\alpha$ level, (F) is the serum IL-1$\beta$ level and (H) is the serum IL-6 level. ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. the control group. *p 0.05 and **p 0.01 vs. the Cisplatin group.

As depicted in Fig. 4E–H, TNF-$\alpha$, IL-1$\beta$, and IL-6 contents were mounted up dramatically in the Cisplatin group (p 0.01). TNF-$\alpha$, IL-1$\beta$, and IL-6 levels were diminished considerably in the HBG and SKP groups (p 0.01 or p 0.05).

NLRP3 and Caspase-1 immunohistochemical staining of kidney tissues ($\times$200), brown is positive expression (Fig. 5A,B). The positive expression of Caspase-1 and NLRP3 in the Cisplatin group was increased substantially. The positive expression of NLRP3 and Caspase-1 in the HBG and SKP groups was reduced dramatically. These findings indicated that HBG diminished the expression of NLRP3 and Caspase-1 in AKI mice.

Fig. 5.

Expression of key proteins in NLRP3/Caspase-1 pathway. (A) NLRP3 immunohistochemical staining of kidney tissues ($\times$200). (B) Caspase-1 immunohistochemical staining of kidney tissues ($\times$200). Western blot results of IL-1$\beta$ and IL-18. (C) is the protein translation of IL-1 $\beta$ and IL-18, (D) is western blot densitometric analysis. ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. the control group. *p 0.05 and **p 0.01 vs. the Cisplatin group.

Results of IL-1$\beta$ and IL-18 Western blots in mouse kidney were displayed in Fig. 5C,D. The expression of IL-18 and IL-1$\beta$ in the Cisplatin group was increased markedly (p 0.01). The expression of IL-18 and IL-1$\beta$ was significantly downregulated in the Cisplatin group. It demonstrated that in AKI mice, HBG decreased the translation of IL-18 and IL-1$\beta$.

The positive translation of TLR4 and NF-$\kappa$B in the Cisplatin group raised substantially (Fig. 6A,B). The positive translation of TLR4 and NF-$\kappa$B in the HBG and SKP groups was reduced dramatically. These detections suggested that in AKI mice, HBG decreased the translation of TLR4 and NF-$\kappa$B.

Fig. 6.

Expression of key proteins in TLR4/NF-$\kappa$B pathway. (A) TLR4 immunohistochemical staining of kidney tissues ($\times$200). (B) NF-$\kappa$B immunohistochemical staining of kidney tissues ($\times$200). Western blot results of IL-6, TNF-$\alpha$ in AKI mice. (C) is the protein translation of IL-6, TNF-$\alpha$, (D) is western blot densitometric analysis. ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. the control group. *p 0.05 and **p 0.01 vs. the Cisplatin group.

Results of Western blot for IL-6 and TNF-$\alpha$ in kidney were displayed in Fig. 6C,D. The translation of IL-6, TNF-$\alpha$ raised massively in the Cisplatin group (p 0.05 or p 0.01). The translation of IL-6, TNF-$\alpha$ in the HBG and SKP groups was dwindled considerably (p 0.01 or p 0.05). It demonstrated that in AKI mice, HBG brought down the expression of IL-6, TNF-$\alpha$.