Study on the protective mechanism of Yizhiren regulating lipid metabolism in mice with diabetic nephropathy

NI Ya-li, YAO Yu-jian, WU Su, XIE Yi-qiang

1.Department of Pharmacy, The Second People’s Hospital of Hainan Province, Wuzhishan 572200, China

2.Medical Department, Hainan Provincial Hospital of Traditional Chinese Medicine, Haikou 570203, China

3.Department of Endocrinology, Hainan Provincial Hospital of Traditional Chinese Medicine, Haikou 570203, China

4.College of Traditional Chinese Medicine, Hainan Medical University, Haikou 571199, China

Keywords:

ABSTRACT Objective: To study the protective mechanism of Yizhiren on the diabetic nephropathy(DN)mouse model based on network pharmacology and metabolomics.Methods: Urea nitrogen,urinary creatinine, urinary albumin were detected in each group of mice, and pathological sections of mice kidney were observed.TCMSP database was used to search for the components and targets of Yizhiren, GeneCards database was used to search for DN-related genes, and drug-component-target-disease network and KEGG signaling pathway were constructed.The fecal samples of mice in the Yizhiren group and the Saline group were collected, metabolomics techniques were used to screen different metabolites, and potential metabolic pathways were analyzed using KEGG database.Results: Compared with the model group, Yizhiren significantly reduced the levels of urea nitrogen, urinary creatinine and urinary albumin in DN mice (P<0.05 or P<0.01), and improved the renal pathology of mice.It was predicted that Yizhiren had 4 effective components and 28 potential targets for treating DN.The KEGG enriched 43 signal pathways, and the main metabolic pathway was lipid metabolism.A total of 584 different metabolites were screened from feces of the two groups (P<0.05).The main metabolic pathways were lipid metabolism, namely sphingolipid metabolism and glycerol phospholipid metabolism, among which the metabolites contained mainly sphingolipin phosphatidylcholine, lysophosphatidylcholine and phosphatidylethanolamine.Compared with the normal saline group, these four metabolites in Yizhiren decreased significantly (P<0.01 or P<0.001).Conclusion: The protective mechanism of Yizhiren in DN mice may be related to the regulation of sphingolipid metabolism, glycerol phospholipid metabolism and the disorder of its metabolites.✉Corresponding author: XIE Yi-qiang, Professor, M.D.

1.Introduction

Diabetes mellitus (DM) is one of the major diseases leading to morbidity and mortality in the world.Diabetic nephropathy (DN)is the most serious renal complication of diabetes mellitus and one of the most important causes of end-stage renal disease (ESRD)[1-3].The molecular mechanism of DN is complex and diverse,which has not been fully elucidated at present[4,5].Yizhiren, sex flavor xin, sex temperature, into the kidney, the spleen meridian, is a good medicine for tonifying the kidney and warming the kidney,at the same time, it is longer than solid astringent shrinkage urine,Xin and not circulating the menstrual flow, warm but not dry, with warm spleen to stop diarrhea salivation, warm kidney shrinkage and solid essence effect.It is commonly used in clinic for enuresis,spermatorrhea and kidney deficiency, etc.It has antioxidant, antitumor, anti-inflammatory and anti-stress effects.Previous studies have found that Yizhiren can significantly reduce blood glucose concentration, catalase, hydrogen peroxide and superoxide anion contents in DN mice, reduce urinary albumin excretion, improve kidney function, and regulate intestinal microbial richness[6,7].

Metabonomics can identify specific molecular markers under specific physiological and pathological conditions, and can be used to study the pathogenesis of metabolic diseases and the action mechanism of therapeutic drugs.Studies have shown that the research based on metabonomics can elucidate part of the mechanism of TCM treatment of DN[8-10].Network pharmacology is a new subject based on the theory of systems biology, and it is one of the new and emphases of modern medical research.It provides abundant information on the bioactive components of single herb or potential prescription and their mechanisms of action at the molecular and system levels[11].Therefore, based on the above,we adopted network pharmacology and metabolomics techniques to screen potential biomarkers and metabolic pathways related to DN in the treatment of Yizhiren, and systematically analyzed the mechanism of action of Yizhiren in the treatment of DN.

2.Materials and methods

2.1 Experimental drugs and animals

Fructus alpiniae Oxyphyllae, was purchased from Hainan Tongrentang Pharmaceutical Company Limited.Irbesartan tablets(Ambovir), purchased from Sanofi (Hangzhou) Pharmaceutical Co.,LTD., lot number: 9A275.Experimental mice (Production license:SCXK(Su)2015-0001, batch No.T002407) were provided by Nanjing Institute of Zoology.

2.2 Reagent

Urea nitrogen test kit (batch number C013) Urine creatinine test kit (batch number C011) All the above reagents were purchased from Nanjing Jiancheng Bioengineering Research Institute.Urinary albumin kit (lot number ml037573), purchased from Shanghai Enzyme Linked Biotechnology Co., LTD.Methanol (CAEQ-4-003302-4000), formic acid (CAEQ-4-014784-0500) and acetonitrile(CAEQ-4-000308-4000) were purchased from CNW Company.L-2-chlorophenylalanine (14091-11-3), purchased from Shanghai Hengchuang Biotechnology Co., LTD.

2.3 Instrument

Glucose meter (Johnson & Johnson, UK); Full-wavelength labeled Instrument (BioDeKInstruments,Inc.); Frozen centrifuge(Eppendorf); Pipette gun (Eppendorf, Germany); Constant temperature water bath (Blue Sky Laboratory Instrument Factory,Hangzhou); Thermostat (Sihao Scientific Instrument, Hubei);Automatic sample rapid grinding instrument (Jingxin Industrial Development Co., LTD., Shanghai); Ultrasonic cleaning machine(Xinzhi Biotechnology Co., LTD., Ningbo); Vortex Oscillator(Hanno Instrument Co., LTD., Shanghai); High resolution mass Spectrometer (Waters); High performance liquid chromatograph(Waters); Chromatographic column (Waters).

2.4 Construction of “Drug-Component-Target-Disease Network Diagram”

Using “Yizhiren” as the search term, the main active constituents and their corresponding targets were retrieved from TCMSP online data (https://old.tcmsp-e.com/tcmsp.php).“Diabetic nephropathy”for the search term, application GenecardS (https://www.genecards.org/) to retrieve genes associated with disease.Using online tools Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) screening of fructus alpiniae oxyphyllae targets for the treatment of DN, and using Cytoscape 3.7.2 software builds “drug - composition - targets -disease network diagram”.

2.5 Pathway enrichment analysis from the Kyoto Encyclopedia of Genes and Genomes (KEGG)

Input the target of Yizhiren in treating DN into Omicshare online workers (https://www.omicshare.com/tools/) to select species “Homo sapiens” to find the pathway of Yizhiren in treating DN and annotate the pathway.

2.6 Drug preparation, mouse grouping and DN mouse modeling

The concentrations of 0.25 g/mL Alpinia oxyphylla decoction and 1.67 mg/mL irbesartan aqueous solution were prepared in laboratory.The mice were divided into four groups, including 8 DB-/DB-mice.24 db-/db-mice were randomly divided into 3 groups with 8 mice in each group.All mice were fed with unlimited water for 12 hours at a temperature of 22 ± 1℃ and a relative humidity of 50 ± 5%.DB-/DB-mice were fed with common diet.db-/db-mice were self-developed type 2 diabetic mice fed with high-sugar and high-fat diet for 4-5 weeks, and the mice were obviously obese and microalbuminuria appeared in 8-9 weeks.DN mouse model was established when fasting blood glucose >16.7 mmol/L (Johnson &Johnson blood glucose meter) and 24 h urinary albumin content 20 mg.DN mice were divided into three groups: Yizhiren group(db-/db-Y), Irbesartan group (db-/db-IRB) and normal saline group(db-/db-S).The intragastric dose of Yizhiren (pre-experiment) and Irbesartan (conversion formula of human and mouse dose: mouse dose = 9.1* human dose mg/kg) are divided into 1.5 mg/g and 0.01 mg/g, and the intragastric dose is about 0.2 mL.Normal group and normal saline group were given corresponding volume of normal saline Continuous intragastric administration for 8 weeks.

2.7 Urea nitrogen, urinary creatinine and urinary albumin were measured according to the kit instructions

2.8 PAS staining

The kidneys were made into paraffin sections →Paraffin section dewaxing →Wash the clear liquid of periodate wine with distilled water for 10 min →After washing with tap water for 10 min,Schiff’s solution staining for 10 min →Rinse with running water for 5 min and stain the nuclei with harisoxylin for 3 min →Rinse with running water for 5 min, conventional dehydration, transparent and sealed

2.9 Pretreatment of fecal samples

2.9.1 Weigh 60mg of feces and add 20 μL of L-2-chlorophenylalanine, 20 μL of Lyso PC17:0, and 600ul of methanolwater (V:V=4:1) to the feces.

2.9.2 Small steel balls were added to the samples Which were placed in a -20 ℃ refrigerator for 2 min for precooling, then in a grinder (60 Hz,2 min) for ultrasonic extraction for 10 min, and then the samples were placed in a -20 ℃ refrigerator for 30 min.

2.9.3 The samples were removed from the refrigerator and placed on a centrifuge at 13 000 rpm, 4 ℃, centrifuged for 10 min.200ul supernatant was absorbed by pipette, filtered by 0.22 μm organic phase pinhole filter, and transferred to LC sample vial for LC-MS analysis.

Remarks: All extracts were pre-cooled at -20 ℃ before use.

2.10 Chromatographic conditions and mass spectrometric conditions

The determination was performed on ACQUITY UPLC BEH C18(100 mm 2.1 mm, 1.7 μm) column.The mobile phases were A-water(containing 0.1% formic acid) and B-acetonitrile (containing 0.1%formic acid).The column temperature was 45℃, the flow rate was 0.4 mL/min, and the sample volume was 2ul.Mass spectrum conditions: electrospray ion source, sample quality spectrum signal acquisition using positive and negative ion scanning mode.

2.11 Statistics

The data of urea nitrogen, urinary creatinine and urinary albumin were statistically analyzed by SPSS16.0 software, and the results were expressed as±s, P < 0.05 was statistically significant.The original data of metabolites were collected with UNIFI 1.8.1.The collected data were processed with SIMCA software package.PLSDA and PCA were used for multivariate statistical analysis of the two groups.Omicshare database (http://www.omicshare.com) was used to visualize P value and Fold change.Through MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) will be screening to the differences of metabolites concentration corresponding metabolic pathways.

3.Result

3.1 Contents of urinary albumin, urea nitrogen and urinary creatinine in mice

After 8 weeks of administration, the contents of urinary albumin,urea nitrogen and urinary creatinine of mice in each group were determined, and the results were shown in Table 1.Compared with the normal group, the contents of urinary albumin, urea nitrogen and urinary creatinine in the normal saline group were significantly increased at 24 h (P < 0.01 or P < 0.01).Compared with normal saline group, the contents of albumin, urea nitrogen and creatinine in urine of irbesartan group and Yizhiren group at 24 h were significantly decreased (P < 0.05 or P < 0.01).

Tab 1 Contents of urinary albumin, urea nitrogen and creatinine in mice of each group(n=8, M/24 h, ±s)

Tab 1 Contents of urinary albumin, urea nitrogen and creatinine in mice of each group(n=8, M/24 h, ±s)

Note: Compared with normal saline group*P <0.05, **P <0.01;Compared to the normal group ##P <0.01, ###P <0.001.

group UA P-value UN P-value UR P-value Normal group 0.09±0.01 - 19.17±1.27 - 5.84±1.93 -IRB group 0.17±0.02** 0.002 29.35±3.36** 0.001 11.78±1.74** 0.003 Yizhiren group 0.38±0.02** 0.003 31.16±4.90* 0.017 9.54±2.01** 0.007 Saline group 1.12±0.04## 0.001 56.89±6.20### 0.000 22.81±2.34### 0.000

3.2 Kidney section

The results were shown in Figure 1.After 8 weeks of administration, compared with the normal group, glomerular diffuse mesangial dilation with mesangial cell proliferation was observed in the normal saline group.Compared with the normal saline group, the mesangial cell proliferation and mesangial dilatation of glomeruli were relatively reduced in Yizhiren group and irbesartan group.

3.3 Construction and analysis of “Drug - component - target- disease network Diagram”

As shown in Figure 2, Yizhiren selected 4 effective components and 28 targets for the treatment of DN.The pink hexagon represents the four active ingredients, which are Daucosterol, sitosterol,Stigmasterol and sitosterol palmitate.The blue circle represents 28 targets, including NFKB1, BAX, PTGS1 and PTGS2.Each activity is linked to multiple targets, and multiple targets are also linked to multiple components.The side surface shows the characteristics of multi-component and multi-target treatment of DN by Yizhiren.

Fig 1 PAS staining of renal pathological section (the arrow is glomerulus)

3.4 KEGG pathway enrichment

Fig 2 “Drug-component-target-disease” network

A total of 43 pathways associated with 28 targets were identified by KEGG enrichment analysis (P<0.05).False positive pathways such as cancer and other diseases with lower P value and top ranking were removed, and KEGG enrichment bubble map ranked top 20 was drawn.The main signaling pathways involved include: NF-kβ signaling pathway, TNF signaling pathway, sphingolipid signaling pathway and cell apoptosis, etc., as shown in Figure 3A.Annotation analysis was carried out on the enriched 43 related pathways,among which lipid metabolism, cofactor and vitamin metabolism,exogenous biodegradation and metabolism as well as metabolism of other substances were related to metabolism, as shown in Figure 3B.Referring to literature[12], we believe that lipid metabolism is one of the main metabolic pathways in the treatment of DN with Yizhien.

Fig 3 Bubble diagram (A) and annotation histogram (B) of KEGG pathways enriched by the potential targets of Yizhien in the treatment of DN

3.5 Preliminary study on metabolites of Yizhien group and DN group

PCA and PLS-DA were used to compare the mass spectrum information of feces of experimental mice.Fecal samples from the model group and Yiyanren group were aggregated respectively in PCA score chart, and the results were shown in Figure 4A.In the PLS-DA figure, the two groups of samples showed good clustering effect, and the two groups were clearly differentiated, and the results were shown in Figure 4B.All the above results indicate that there are metabolic differences between the two groups of samples

Fig 4 PCA(A) and PLS-DA (B)

3.6 Analysis of different metabolites between Yizhiren group and DN group

According to the conditions of VIP >1 and P < 0.05 in t test, a total of 584 potential differential metabolites between Yiyanren group and DN group were screened by OPLS-DA analysis.Including phosphatidylcholine PC(16:0/0:0), LysoPC(18:1(9Z))and phosphatidylcholine PG(21:0/0:0).The results are shown in Figure 5.According to the weight value of variable (VIP value),the expression of differential metabolites in the top 30 pairs with VIP value was visualized.The larger the VIP value, the greater the influence of the metabolite in the two groups.

Fig 5 Heatmap of differential metabolites in the top 30 VIP values

3.7 Analysis of metabolic pathways of different metabolites in Yizhiren group and saline group

The MetaboAnalyst 5.0 online database was used to construct corresponding metabolic pathways for differential metabolites, and metabolic pathways with impact value > 0.10 and -log(P) > 1.5 were selected as potential target metabolic pathways.

The results were shown in 6, where the influence values of sphingolipid metabolism and -log (P) values were 0.154 and 2.355,and glycerol phospholipid metabolism and -log (P) values were 0.216 and 1.697, indicating that the metabolic disorder of DN mouse model was mainly related to sphingolipid metabolism and glycerol phospholipid metabolic pathway.Combined with the results of differential metabolites analysis, the metabolites of sphingolipid metabolism and glycerol phospholipid metabolism pathway mainly include sphingolipin, phosphatidylcholine, lysophosphatidylcholine and phosphatidylglycolamine.Compared with the saline group,the levels of these four metabolites in the Yizhiren group were significantly decreased (P < 0.01, P < 0.001), and the results were shown in Figure 7.

Fig 6 (DN) related metabolic pathways

Fig 7 The content difference of four metabolites in two groups

4.Discuss

Diabetic nephropathy is characterized by progressive renal dysfunction and glomerular filtration rate (GFR)<60 mL/ min /per1.73 m2.The routine diagnosis methods of DN mainly include increased urinary albumin excretion rate, decreased glomerular filtration rate and renal pathological changes.In this study, the therapeutic effect was evaluated by measuring the contents of urinary albumin, urinary creatinine and urea nitrogen in urine at 24 hand observing the pathological changes of kidney.It was found that Yizhiren can significantly reduce the 24 h urine protein, urinary creatinine and urea nitrogen excretion, and reduce mesangial cell proliferation and mesangial dilatation of model mice.

The results of “drug-component-target-disease network diagram”showed that 28 genes related to DN were NR3C1BAXKL and PTGS2, respectively.The Glucocorticoid receptor (NR3C1) is mainly located in glomeruli, proximal tubules and coarse rising branch of ansa medulla, and is present in endothelial cells,podocytes, mesangial cells and endothelial cells.Studies have shown that endothelial NR3C1 deficiency accelerates the development of diabetic nephropathy, and podocyte NR3C1 is crucial for glomerular endothelial cell homeostasis in diabetic patients[13,14].

Proapoptotic protein (Bax), a member of the Bcl-2 family, is a core regulator of the intrinsic pathway of apoptosis.The expression level of Bax is significantly up-regulated in podocytes induced by high glucose[15].KL (Klotho) is an anti-aging molecule, which is expressed in the choroidal plexus of the main kidney and brain.Overexpression of KL may have a potential therapeutic effect on the treatment of diabetes and diabetic nephropathy[16].PTGS2, also known as cycloxygenase-2 (COX-2), is a key gene in the synthesis of prostaglandins from arachidonic acid.Singh B, et al.found that inhibition of upregulation of COX-2 could improve the disease status of diabetic nephropathy rats[17].

The results of KEGG pathway enrichment analysis showed that lipid metabolism was one of the main metabolic pathways in the treatment of DN with Yizhiren.The metabolic pathways of sphingolipid metabolism and glycerol phospholipid metabolism revealed by metabonomics also confirm this view.Based on the analysis of different metabolites between Yizhiren group and DN group, the metabolites contained in the above metabolic pathway were mainly Sphingomyelin phosphatidylcholine lysophosphatidylcholine and phosphatidylethanolamine, and the contents of these four metabolites decreased significantly after the administration of Yizhiren in mice of model group.Metabolites change with the progression of kidney injury and become new sensitive biomarkers.Sphingomyelin is one of the main components of eukaryotic biofilm phospholipids,which together with phosphatidylcholine constitute more than 50%of membrane phospholipids.Sphingomyelin has been reported to regulate inflammation, adipose tissue function, obesity, diabetes, and the development of related diseases such as atherosclerosis[18,19].Studies have shown that high sphingomyelin levels may contribute to the onset and progression of kidney disease and increase the risk of end-stage renal disease[20].Phosphatidylcholine is the main component of cell membrane, the source of lipid second messenger and the determinant of cell cycle progression.Phosphatidylcholine has many biological activities in vivo, such as anti-inflammatory[21],improving brain function[22] and regulating lipoprotein homeostasis[23].It was found that the content of phosphatidylcholine in diabetic nephropathy group was significantly higher than that in normal group[24].Lysophosphatidylcholine is a lipid biomolecule formed by the lysis of phosphatidylcholine, which has harmful effects on many kinds of cells through G protein-coupled receptor pathway, including enhancing inflammatory reaction and inducing apoptosis and insulin resistance[25].The rapid progression of renal dysfunction in diabetic nephropathy is associated with increased lysophosphatidylcholine, which induces lipid droplet accumulation through the activation of peroxisome proliferator-activated receptor, resulting in up-regulation of lipid droplet coated protein Perilipin 2 and decreased autophagy flux, leading to proximal renal tubule organelles stress and apoptosis[26].Phosphatidylethanolamine is the second most abundant glycerol phospholipid in eukaryotic cells.It is functionally related to protein biogenesis and activity, oxidative phosphorylation, autophagy, membrane fusion and mitochondrial stability, and is an important precursor of other lipids.It is found that phosphatidylethanolamine is a metabolite that promotes early diabetic kidney damage, the levels of phosphatidyl ethanolamine in the glomeruli and renal tubules were significantly increased in diabetic patients compared to non-diabetic controls [27].

In summary, this study adopted network pharmacology combined with metabolomics technology to further reveal the effectant substances, target sites, metabolites and metabolic pathways of Yizhiren in the treatment of diabetic nephropathy, fully reflecting the multi-component, multi-target and multi-pathway action characteristics of Yizhiren in the treatment of diabetic nephropathy.This method provides a scientific basis for the future study of Yizhiren in the treatment of DN.

Description of contributions of all authors: Ni Yali was responsible for experimental design, data processing and article writing; Yao Yujian and Wu Su are responsible for TCM theory interpretation,animal modeling, sample collection and testing; Xie Yiqiang was responsible for the experimental ideas and the final revision of the paper.

Disclaimer: All authors have no conflict of interest in this article.