Effects of Huangqi Liuyi Decoction in the Treatment of Diabetic Nephropathy and Tissue Distribution Difference of its Six Active Constituents Between Normal and Diabetic Nephropathy Mouse Models

The purpose of this study was to investigate the effects of Huangqi Liuyi decoction extract (HQD) on diabetic nephropathy (DN), and the tissue distribution difference of six main active ingredients of HQD between normal and DN mouse models. DN mice were administered HQD for 12 weeks to investigate its efficacy in the treatment of DN. Liquid chromatography-tandem mass-spectrometry (HPLC-MS/MS) was used to analyze the tissue distribution of the six active ingredients of HQD in normal and DN mice, including astragaloside IV, calycosin-7-O-β-D-glucoside, calycosin glucuronide, ononin, formononetin, and glycyrrhizic acid. DN mice treated with HQD showed significantly decreased fasting blood glucose (FBG), 24-h urinary protein (24 h U-Alb), blood urea nitrogen (BUN), serum creatinine (Scr), and triglyceride levels (TG) (p < 0.05). Moreover, there were no significant differences in pharmacodynamics between HQD and Huangqi Liuyi decoction. Treated mice also had decreased expression of collagen I, ɑ–smooth muscle actin (ɑ-SMA), and vimentin; and upregulated expression of E-cadherin in their kidneys. Compared to normal mice, distributions of the six ingredients in the liver, heart, spleen, lungs, kidneys, stomach, small intestine, brain, and muscle of DN mice were different. The results indicated that the HQD could be used for the treatment of DN and to improve renal function. The pathological state of diabetic nephropathy may affect tissue distribution of HQD active ingredients in mice.


INTRODUCTION
Diabetic nephropathy (DN) is an irreversible condition characterized by a continuous decline in the glomerular filtration rate, proteinuria, microalbuminuria, and increased blood pressure (Xiao et al., 2008;Nowak et al., 2018;Kopel et al., 2019). Most individuals with DN progress to end-stage kidney disease (Noor et al., 2020). Prevention or early treatment of DN lowers treatment costs and improves the survival rate and quality of life of patients (Correa-Rotter and Gonzalez-Michaca, 2005). Traditional Chinese medicines (TCMs) have been applied in the clinical treatment of various diseases (Peng et al., 2020). TCMs offer unique advantages in the prevention of diabetic complications because of their limited side effects and/or reduced toxicity (Shi et al., 2011;Sun et al., 2016). Huangqi Liuyi decoction has been used in China since the Song dynasty. It is composed of Radix Astragali and Radix Glycyrrhizae. Astragali inhibits the formation of kidney interstitial fibrosis and retards the development of diabetic nephropathy. Glycyrrhizae decreases fasting blood-glucose and kidney oxidative stress (Lu and Wei, 2014;Ma et al., 2019). Huangqi Liuyi decoction significantly decreased fasting blood glucose levels and kidney damage in diabetic rats (Xu et al., 2017;Wen et al., 2018). However, precise and reliable dosing with TCMs remains challenging, which negatively impacts the reproducibility of research and clinical results. Identification of the active constituents of a given compound and ensuring consistency in formulation may overcome the problem with TCM variability (Zhang and Wang, 2005). Relative to DN, our team found that the main active constituents of Huangqi Liuyi decoction were astragalus saponin, astragalus flavone, astragalus polysaccharide, and glycyrrhizic acid (data unpublished). Herein, astragalus saponin, astragalus flavone, astragalus polysaccharide, and glycyrrhizic acid were recombined into mixed extract (HQD), and its effect on DN was determined.
In recent years, ample research has shown that the absorption, tissue distribution, and metabolism of drugs can be affected by the disease state. The pharmacokinetic characteristics in pathological conditions are different from those in the normal condition in a manner directly related to the efficacy and adverse reactions of drugs (Shang et al., 2017;Yang and Liu, 2019;Zhang et al., 2020). Thus, it is necessary to compare the tissue-distribution characteristics of drugs under both normal and pathological conditions. In the present study, a liquid chromatography-tandem massspectrometry (HPLC-MS/MS) method was established to investigate differences in the distribution of HQD in tissues and organs under normal and pathological conditions. This study provides additional insights into the safe usage of HQD, and TCMs in general, in healthy mice and those with diabetic nephropathy.
Rosiglitazone ( A mixture of four active constituents (HQD) from Huangqi Liuyi decoction was self-made. In the early stage, the research group determined the preparation process of astragalus saponins, astragalus flavones, astragalus polysaccharides, and glycyrrhizic acid extract. Astragalus saponins and astragalus flavones extract were prepared by macroporus resin column, astragalus polysaccharides extract was prepared by water extraction and alcohol precipitation, and glycyrrhizic acid extract was prepared by acid precipitation. The extract content of each component extracted from three batches of Astragalus and Glycyrrhiza is shown in Table 1.

Determination of Biochemical Indexes
Male db/db mice aged 12 weeks were randomly divided into 6 groups of 6 mice each. Twelve-week-old db/m mice served as a control group. The clinical crude drug dosage of Huangqi Liuyi decoction was Astragalus 60 g/d and Glycyrrhiza 10 g/d, with a total dose of 70 g/d (Xv et al., 2017). Astragalus and Glycyrrhiza were decocted 3 times, the extract was concentrated and dried, and the dosage of db/db mice was converted according to the above clinical dosage of the drug. The dosage of Huangqi Liuyi decoction was 10.62 g/kg. Mice treated with rosiglitazone (0.61 mg/kg) and valsartan (12.13 mg/kg) served as a positive control group. The doses of HQD in high-, medium-, and low-dose groups were 0.96 g/kg, 0.48 g/kg, and 0.24 g/kg, respectively, which were 4 times, 2 times, and 1 times the clinical dosage. Each group was treated daily for 12 weeks. The control group was given the same volume of distilled water. All mice were fasted for 12 h before the experiment, the FBG level was measured. In addition, 24-h urine was collected, and the 24-h U-Alb level   Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 934720 5 was determined using a Coomassie brilliant blue quantitative method (Nie et al., 2019). The BUN, Scr, TG, and TC values were determined using a standard biochemical apparatus (Roche, cobacc501, Switzerland). Subsequently, the mice were humanely euthanized and their kidneys harvested.

Histopathology Analysis of Kidney Tissues
Paraffin-embedded kidney specimens were sectioned into slices with a thickness of 3 μm using a microtome. After deparaffinization and rehydration, the slices were stained with hematoxylin and eosin for the assessment of kidney injury and with Masson's trichrome staining for a collagen-deposition assessment. The slices were observed under a light microscope, and images were captured at a magnification of ×400 using the objective lens of an Olympus microscope (Olympus Corporation, Tokyo, Japan). The light microscopy evaluation was conducted by experienced pathologists in a blinded fashion.

Immunohistochemical Assay
Kidney tissue sections (3-μm thickness) were deparaffinized with xylene and rehydrated in a gradient ethanol finishing in phosphate-buffered saline. Endogenous peroxidases were quenched by a few drops of H 2 O 2 . A citrate buffer solution was used to restore the antigens, and the kidney tissues were then sealed with goat serum. After the sections were incubated with the primary antibody overnight, the secondary antibody was applied. Then, the sections were washed with phosphate-buffered saline, dminobenzidine (DBA) was added for color rendering, and counterstaining was completed with hematoxylin. A positive expression was indicated by a brownish-yellow color. For immunohistochemical staining, the average integrated positive area from 6 randomly chosen regions was calculated by using Image Pro Plus 6.0 image analysis software.

Statistics
All data are presented as mean ± standard deviation values. Statistical analysis between the 2 groups was performed using the Statistical Package for the Social Sciences version 23 software program (IBM Corporation, Armonk, NY, United States). p ≤ 0.01 and p ≤ 0.05 between the 2 groups were considered statistically different.

Tissue Sample Preparation
Tissues were accurately weighed and homogenized 4 times with normal saline. Then, 200 μl of tissue homogenate and 20 μl of IS solution (60.9 ng/ml of puerarin and 4.02 μg/ml of digoxin) were added into 400 μl of methanol-acetic acid (40:1, v/v). Samples were vortexed for 2 min and centrifuged at 6,000 r/min for 10 min. The supernatant was transferred and evaporated to dryness with a nitrogen-blowing instrument (Organomation, Berlin, MA, United States) at 37°C, and the residue was sonicated with 200 μl of 50% methanol and centrifuged for 10 min at 10,000 rpm. One microliter of supernatant was injected into the HPLC-MS/MS system for analysis. Quality control samples were prepared separately in the same way.

Conditions of HPLC-MS/MS
An Acquity HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a Q-Trap ® 5500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, United States) was employed for HPLC-MS/MS. The chromatographic conditions of the six constituents of HQD were determined using an Excel2C18-AR system (100 × 2.1 mm, 2 μm; Advanced Chromatography Technologies Ltd., Aberdeen, Scotland) maintained at 30°C. Analysis was completed with a gradient elution of 0.1% formic acid (A) and acetonitrile (B) and a flow rate of 0.4 ml/min. The gradient elution was as follows: 0-0.6 min (90% A), 0.6-2 min (90%→70% A), 2-6 min (70%→35% A), 6-8 min (35%→10% A), 8-9 min (10%→10% A), 9-9.1 min (10%→90% A), and 9.1-12 min (90% A). For MS/MS detection, an electrospray ionization in a multi-reaction monitoring mode was operated with polarity switching between negative and positive ion modes. The mass spectrometer parameters were set as follows: ion spray voltage at 5.5 kV (+) and −4.5 kV (−), source temperature at 600°C, nebulizer pressure at 55 psi, curtain gas at 30 psi, and auxiliary gas Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 934720   Figure 1 shows the chemical structure of six analytes.  The specificity of the method was evaluated by analyzing homogenates of drug-free tissue, homogenates of drug-free tissue containing standard solutions and IS, and homogenates obtained following oral administration of HQD to check whether the determination was interfered with by endogenous substances.

Calibration Curves and Linearity
The linearity of the calibration curve was constructed using eight calibration points for tissue homogenate. Briefly, peak area analyte/IS ratios (Y) were tested against the theoretical concentration of each analyte (X) using 1/X 2 weighting of the linear regression. The lowest concentration in the calibration curve was defined as the lower limit of quantification.

Accuracy and Precision
The quality control samples at three concentration levels of analytes were prepared and operated in parallel according to the above methods of sample preparation. Each concentration was analyzed during six replications. Assay  Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 934720 precision was calculated using relative standard deviation (RSD, %) and variance, and accuracy is expressed as mean ± standard deviation.

Extraction Efficiency and Matrix Effect
The recovery and matrix effect of the analytes were analyzed for medium concentration quality control samples. The extraction recovery was determined by comparing the response ratio of extracted samples with extracted blank matrix spiked with corresponding concentrations. The matrix effect was evaluated as the peak area ratio of analytes spiked with blank tissue extract at medium concentrations to non-extracted QC standard solutions at equivalent concentrations.

Stability
Quality control samples at three concentration levels of tissue samples were prepared to investigate the stability of tissue samples. First, we stored samples at room temperature (approximately 25°C) for 24 h, and then froze (−20°C) them for 48 h and repeated this freezing and thawing cycle three times. The samples were processed based on the abovementioned sample processing method and then measured by HPLC-MS/MS.

Tissue Distribution
Thirty-two db/db model mice and 32 normal db/m mice were fasted for 12 h. Then, the diabetic and normal mice were randomly divided into 4 groups of 8 mice each. One group of mice served as a unique sampling time point. Mice were given a single dose of HQD (high dosage in efficacy study). Blood was collected by retro-orbital venous puncture at 30 min and 2, 4, and 8 h. Subsequently, the mice were humanely euthanized, and the heart, liver, spleen, lungs, kidneys, stomach, small intestine, brain, and skeletal muscle were collected. Samples were washed with normal saline and stored at −20°C. Before the experiments, the tissues were prepared according to the sample-preparation methods and analyzed by HPLC-MS/MS.

Effect of Huangqi Liuyi Decoction on Diabetic Nephropathy
After 12 weeks, the weight of the model group was significantly decreased, the hair had lost its normal luster and was sparse, and the animals appeared less active. Compared with before administration, the weight of the HQD administration group was not significantly decreased, their fur remained shiny, and the animals were active (Figure 2). Compared to those of the control group, 12-week-old diabetic mice showed increased FBG, BUN, Scr, TG, TC, and 24 h U-Alb levels (p < 0.05) (Figure 2). Diabetic mice treated with HQD showed lower biochemical parameters compared to untreated (p < 0.05). There was no significant difference in parameters between HQD and Huangqi Liuyi decoction treated animals (p > 0.05).
Masson's trichrome stained collagen fibers blue, while muscle fibers, cytoplasm, cellulose, and keratin appeared red. Kidney sections from of 12-week-old db/db diabetic mice showed increased collagen fibers in the glomerular basement In non-diabetic control mice, the glomerular structure showed no pathologic changes in the glomerular mesangial area ( Figure 4). Conversely, kidneys from diabetic mice showed mesangial matrix deposition and thickening and nodular sclerosis of the glomerular capillary basement membrane. Diabetic mice treated with HQD showed less kidney damage compared to untreated.

Protein Expression of Collagen I, E-Cadherin, ɑ-Smooth Muscle Actin, and Vimentin
The semi-quantitative analysis of collagen I, E-cadherin, ɑ-SMA, and vimentin is showed in Figures 5-8. Expression of collagen I, ɑ-SMA, and vimentin was significantly increased (p < 0.01) and that of E-cadherin was significantly decreased (p < 0.01) in kidney sections from diabetic mice compared to kidney samples from non-diabetic mice. In contrast, diabetic animals treated with HDQ or decoction showed less kidney collagen I, ɑ-SMA, and vimentin (p < 0.05); and more E-cadherin (p < 0.01) versus samples from untreated diabetic mice. Chromatograms of the blank tissue homogenate (e.g., heart, lung, and renal); blank tissue homogenate spiked with astragaloside IV, calycosin-7-O-β-D-glucoside, calycosin-glucuronide, ononin, formononetin, glycyrrhizic acid, and IS; and tissue homogenate obtained after oral administration of HQD are displayed in Figure 9. Good separation was observed among the ingredients, and there was no interference from the endogenous substances in the determination of the six ingredients and IS.

Calibration Curves and Linearity
The typical equation of linearity ranges and calibration curves for the six ingredients are shown in Table 2 (e.g., heart, lungs, and kidneys). The lowest concentration in the calibration curve was defined as the lower limit of quantification. The results show that all correlation coefficients are higher than 0.999, indicating that the concentrations of the six ingredients in mouse tissues correlated well within the linearity ranges. Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 934720

Accuracy and Precision
Intraday and interday precision and accuracy (e.g., heart, lungs, and kidneys) are summarized in Table 3. The intraday and interday RSDs were below 20.0%. Thus, the accuracy and precision results met the requirements of biological sample detection.

Extraction Efficiency and Matrix Effect
Results of the extraction efficiency and matrix effect analyses are shown in Table 4 (e.g., heart, lungs, and kidneys). As noted, the recoveries of the six ingredients were precise, consistent, and reproducible at different concentration levels in various tissue samples, and there was no significant matrix interference.

Stability
Results of the stability analysis are shown in Table 5 (e.g., heart, lungs, and kidneys). The stability test results indicated that the mouse tissue samples showed good stability under the three different conditions with a 10% concentration variation compared to the initial values.

Tissue Distribution Study
Following a single oral administration of HQD, the distribution of the six ingredients (astragaloside IV, calycosin-7-O-β-D-glucoside, calycosin glucuronide, ononin, formononetin, and glycyrrhizic acid) between the normal and DN mouse groups were analyzed at different time points (30 min and 2, 4, and 8 h). The six ingredients were detected in all tissue samples from diabetic and nondiabetic mice (Figures 10-(15). Astragaloside IV, calycosin-7-O-β-D-glucoside, calycosin glucuronide, ononin, formononetin, and glycyrrhizic acid were found highest in the lungs and kidneys. However, the six active ingredients were minimally found in the brain. As expected, tissue distribution of all ingredients decreased with time. Compared with the normal group, astragaloside IV levels in the hearts, kidney, and brains of mice with DN were higher at 30 min; levels of astragaloside IV in the livers, lungs, intestines, and muscles of mice with DN were higher at 2 h. Astragaloside IV levels in the hearts, kidneys, intestines and brains of mice with DN were higher at 4 h. Levels of astragaloside IV in the hearts, livers and kidneys of mice with DN were higher at 8 h ( Figure 10).
Compared with the normal group, DN mice had higher levels of calycosin-7-O-β-D-glucoside in their hearts, spleens and intestines at 30 min. Calycosin-7-O-β-D-glucoside levels in the lungs and kidneys of DN mice were higher at 2 h. Levels of calycosin-7-O-β-D-glucoside in the hearts, livers, spleen, and kidneys of DN mice were higher at 4 h ( Figure 11).
DN mice had higher levels of calycosin-glucoside in their livers, kidneys, and stomachs than the normal group at 30 min. Levels of calycosin-glucoside in the hearts, spleens, lungs, and kidneys of mice with DN were higher at 2 h ( Figure 12). Ononin levels were higher in the spleens, lungs, kidneys, and intestines of DN mice at 30 min, compared with the normal group. DN mice had higher levels of ononin in their hearts at 2 h. Levels of ononin in their hearts, lungs, stomachs, and muscles were higher at 4 h. Ononin levels in the kidneys and stomachs were higher at 8 h ( Figure 13).
Compared with the normal group, DN mice had higher levels of formononetin in their hearts, livers, spleens, lungs, and kidneys at 30 min. Formononetin levels were also higher in their stomachs and intestines at 2 h. Levels of formononetin in the hearts, livers, kidneys and intestines of DN mice were higher at 4 h ( Figure 14).
Compared with the normal group, glycyrrhetinic acid levels were higher in the heart, spleens, and lungs of mice with DN at 30 min. DNs had higher levels of glycyrrhetinic acid in their hearts and kidneys at 4 h. Levels of glycyrrhetinic acid in the hearts, kidneys, and intestines of mice with DN were higher at 8 h ( Figure 15).

DISCUSSION
In this study, we used 12-week-old db/db mice as a diabetic nephropathy mouse model and 12-week-old db/m mice as a normal comparison group. The db/db mouse was a mutant type, with the line of leptin receptor gene defect picked from C57BL/6J mice by the Jackson Laboratory of the United States. The db/db mouse spontaneously develops type 2 diabetes and is similar to clinical type 2 diabetes. db/db mice display obesity, hyperlipidemia, and hyperglycemia after 4 weeks of age. Diabetic nephropathy is found after 8-12 weeks of age (Gerald et al., 2013;Ponchiardi et al., 2013). The experimental results showed that the FBG, Scr, BUN, TC, TG, and 24 h U-Alb of 12week db/db mice were significantly higher than those of the normal control group (p < 0.05). The kidneys of 12-week db/db mice also showed obvious glomerular lesions.
Some researchers have investigated in vivo content analysis methods for Huangqi Liuyi decoction (Zeng et al., 2018), but a method for simultaneously quantifying various ingredients in multiple organs has not yet been reported. In this research, HPLC-MS/MS was used to analyze the tissue distribution of the six active ingredients of HQD in normal and DN mice. Furthermore, the methodology, including specificity, linearity, accuracy, precision, extraction efficiency, matrix effect, and stability, was verified. The content of drugs in tissues and organs is usually difficult to measure, in part because there are many factors that interfere with the process. Triple quadrupole mass spectrometry is often used to determine trace components in biological samples, such as blood, urine, and tissue, in pharmacokinetic studies. In its multi-reaction monitoring scanning mode, the precursor ions of the tested component are prescreened in the first quadrupole (Q1), separated in the second quadrupole (Q2), and identified and quantified in the third quadrupole (Q3). In comparison with the traditional triple quadrupole mass spectrometry approach, the QTRAP ® LC-MS/ MS system (Sciex, Framingham, MA, United States) is equipped with an additional linear ion trap at the third quadrupole (Q3). This new generation of HPLC-MS/MS system, which is equipped with a