Rui Kai, Wang Xian, Zhao Jing-Yu, Ma Tian-Peng, Jiang Huan, Yang Xu, Xie Yi-Qiang, Li Kai

1. Hainan Medical University, Haikou 571199, China

Keywords:Diabetic kidney disease Tonifying kidney and dispelling pathogenic wind Alpinia Oxyphylla ; Saposhnikovia Radix Network pharmacology

ABSTRACT Objective: Network pharmacology was used to predict the main targets and related mechanism pathways of the key compounds and active ingredients of the Alpinia oxyphylla-Saposhnikovia Radix based on the therapeutic strategy of tonifying kidney and dispelling pathogenic wind for the treatment of diabetic kidney disease(DKD). Methods: The effective components of Alpinia oxyphylla-Saposhnikovia Radix were screened through the data platform of Traditional Chinese Medicine(TCM). After screening effective targets, the disease target gene database was used to obtain targets for DKD, and then gene enrichment analysis to predict relevant signalling pathways after obtaining the key target genes through Venn analysis. Results: There were 324 active components of Alpinia oxyphylla-Saposhnikovia Radix through the data platform of TCM. In addition, Venn analysis showed that there were 48 key target genes for the treatment of DKD.The biological pathways for the action of Alpinia oxyphylla-Saposhnikovia Radix in DKD were closely related to many signaling pathways including NF-kappaB, classical MAP kinase, JNK and P38 MAP kinase, and ERK5. Conclusions: Treatment of DKD with Alpinia oxyphylla-Saposhnikovia Radix based on the therapeutic strategy of tonifying kidney and dispelling pathogenic wind has the characteristics of multi-components, multi-targets and multi-pathways,which could provide new ideas and methods for further researches on the treatment of DKD with Alpinia oxyphylla-Saposhnikovia Radix.

1. Introduction

Diabetic kidney disease(DKD) is one of the most important microvascular complications of diabetes, and it is the main cause of end-stage-renal-disease(ESRD)[1].According to epidemiological statistics, there will be about 464 million diabetes patients in the world by 2019, by 2045, the number of people with diabetes is expected to exceed 700 million [2]. After 10-20 years of diabetes,about 20% - 40% of patients will develop DKD, The final progress is ESRD [3]. In chronic kidney disease, the mortality rate of DKD increased gradually, DKD has become the first reason for ESRD [4].At present, the main strategies of Western medicine in the treatment of DKD are the control of blood glucose and blood pressure and inhibit RAAS.Although SGLT2 inhibitors and other new drugs have shown therapeutic effect on DKD in recent years, their adverse reactions and worsening renal end-point events limit their clinical application [5].

The method of tonifying the kidney and dispelling wind is a common method in the clinical treatment of DKD in Traditional Chinese medicine (TCM), which was determined by Professor Guo Ziguang, the first master of traditional Chinese medicine, aiming at the pathogenesis of "kidney deficiency and wind" in DKD.[6]. Yi Zhiren(Alpinia oxyphylla, AO)is one of the four important southern Chinese Herbal medicines(CHM) in Hainan. It has warm nature and pungent taste, enters the spleen and stomach meridian, and has the effect of warming the spleen and relieving diarrhea, taking saliva, warming the kidney, fixing essence and reducing urine. The property-flavor of Fangfeng (Saposhnikovia Radix, SR ) is lukewarm,pungent and sweet. It can enter the meridians of bladder, lung,spleen and liver. It has the effect of dispelling wind and relieving surface, winning dampness and relieving pain, antispasmodic effect.AO-SR is a representative CHM combination for tonifying kidney and expelling pathogenic wind. They can effectively target the pathogenesis of kidney deficiency and wind evil in DKD [7-8]. In the previous study of the team, it has been proved that AO is effective in the treatment of DKD, such as inhibiting inflammatory pathway,protecting renal function, reducing trace protein leakage, improving pathological changes of diabetes, etc.,[9-10]and confirmed that its specific mechanism is to intervene in NLRP3 inflammatory signal pathway and inhibit the expression of NLRP3 inflammatory factors to reduce renal inflammatory response.The specific mechanism of NLRP3 is to reduce the expression of NLRP3 inflammatory factors by inhibiting the NLRP3 inflammatory pathway, thereby reducing the renal inflammatory response[11].At the same time,we have confirmed the efficacy of AO-SR to treat DKD through pharmacodynamic experiments, but its specific active ingredients and mechanism of action need further in-depth study.The method of tonifying kidney and expelling pathogenic wind is an effective method to treat DKD under the guidance of the whole concept of TCM. However, in the current studies on the treatment of DKD by AO-SR, the effective ingredients and mechanism of action are still unclear, and it is difficult to study comprehensively and systematically from the whole to the molecular level.The mechanism of "multi-component, multi-target, multi-channel" in the whole research idea of network pharmacology system is consistent with the overall concept of TCM and the concept of drug compatibility and efficiency [12]. At the same time, the incidence of DKD is related to many factors [13].Based on network pharmacology, the research on the mechanism of AO-SR on "multi-component, multi-target, multichannel" can provide more research ideas and programs for the application of AO-SR and the treatment of DKD.

2. Method

2.1 Screening of potential active ingredients of AO and SR and searching for corresponding target proteins

In the TCM data platform, AO and SR were used as the key words to search the main compounds, and the screening indexes were OB＞ 30% and DL ＞ 0.18; and the effective targets were selected by combinescore ≥ 0.5 through the target gene prediction platform.

2.2 Screening of DKD related targets

In the database of disease target genes, with diabetic kidney disease as the key word, proteins with binding score ＞ 0.8 were selected as specific target genes.

2.3 Screening and pathway prediction of target genes of AOSR and DKD

The component target and specific target genes were analyzed by Venn analysis, and the key target genes were obtained. Then the protein interaction platform was used to predict the pathway

2.4 Related software and data platform used in this paper

The main ingredients of traditional Chinese medicine and their prediction databases are:NPASS(http://bidd2. nus.edu. sg/NPASS)、HIT(http://lifecenter. biosino. org/hit/)、BATMANTCM(http://bionet.ncpsb. org/batman-tcm/)、TCMSP(http://lsp. nwu.edu. cn/tcmsp. php)、PubChem(https://pubchem.ncbi.nlm.nih.gov/)；The target genes prediction database mainly used are:STITCH(http://stitch.embl.de/)、Swisstarget prediction(http://swisstarget prediction.ch/)、TargetNet(http://targetnet.scbdd.com)；The database of protein interaction platforms mainly used are:STRING(https://string-db.org/)、GeneMANIA (http://genemania.org/)；DAVID(https://david. ncifcrf.gov/)、Cytoscape 3.6.1(http://www.cytoscape.org)、OMICshare (https://www.omicshare.com/)Used to build analytical interaction networks.

3. Result

3.1 Screening of effective chemical constituents and effective targets of AO and SR(STITCH、Swisstarget prediction、TargetNet)

Use the traditional Chinese medicine data platform to search the main ingredients of AO and SR:

(1) AO:Stigmasterol, Daucosterol, sitosterol, sitosterol palmitate.

(2) SR:PENTACOSANOIC ACID、2-[(2S)-7-oxo-2,3-dihydrofuro[4,5-g]chromen-2-yl]propan-2-yl (E)-2-methylbut-2-enoate、heptadeca-1,8-dien-4,6-diyn-3,10-diol、(2R,3R)-3-(4-hydroxy-3-methoxy-phenyl)-5-methoxy-2-methylol-2,3-dihydropyrano[5,6][1,4]benzodioxin-9-one、anomalin、divaricatacid、11-hydroxy-sec-o-beta-d-glucosylhamaudol_qt、ledebouriellol.

Screening conditions:OB＞30%、DL＞0.18.

Target gene prediction platforms (STITCH, Swisstarget prediction,TargetNet) were used to predict target genes of effective compounds or analogs on-line, and 124 effective targets were screened by setting condition combinescore 0.5.

3.2 Mining related genes

We used the target gene database of diabetic nephropathy disease to search the target protein that have been confirmed to be included in the database.The original data were collected,and selected the protein with binding score ＞ 0.8 as the specific target gene. 301 targets were obtained. The results are as follows. Some results are shown in Tab 1

Tab 1 Specificity targeted gene list

3.3 Network construction analysis (enrichment)

The component targets and specific target genes screened in the first two steps were analyzed by Venn analysis, and 36 key target genes were obtained: MLK、MKK4、MEK1、MEK2、NF-kB、AKT、CDK2、HBXAP、NLK、MB、GSK3B、HLA-DPB1、PIK3R5、CDK6 and so on.

Figure 1 The Key Compound

The selected effective compound targets correspond to the compounds to which they belong, and the relationship between them was constructed. After entering the file into the Cytoscape software for mapping, the results were found after Hub Analysis: heptadeca-1,8-dien-4,6-diyn-3,10-dio, divaricatacid, 11HYOMAUTOL QT.The results are shown in Figure 1.The common gene data were collected and imported into protein interaction platform (STRING、GeneMANIA) to extract the interaction relationship. The file was saved and entered into Cytoscape software for mapping. After hub analysis, it was found that GSK3B、ILF2、STAT3、CDK1、ESR2、SHC1 were in the central position of association, which may be the key target genes.The results are shown in figure 2.

Figure 2 The Key Targeted Gene

3.4 Gene enrichment analysis

The obtained key target genes were recorded into GO functional enrichment and KEGG enrichment of key genes in David database. The top related signal pathways are as follows: JNK、Cell Apoptosis、PBX3、ERK5 pathway, etc. After target gene mapping signal pathway, The results obtained are concentrated in inflammatory or immune pathways such as NF-kappaB、classical MAP kinase、JNK and p38 MAP kinase、ERK5 and so on.The results are shown in the figure 3.

Figure 3 The critical pathway

4. Discussion

Tonifying kidney and dispelling pathogenic wind method has definite clinical effect in the treatment of DKD.The pathogenesis of"kidney deficiency" is involved in the whole process of DKD. The pathogenesis of kidney deficiency in DKD has been recorded in ancient medical books. Synopsis of the Golden Chamber records that"When a person has diabetes, however, more urine will be urinated,and the water he drinks is quickly drained out of his body. It can be treated with Shenqi pill". This discusses DKD kidney deficiency causes thirst, frequency of urination. The General Medical Collection of Royal Benevolence records that "If the course of diabetes is long,the kidney qi will be injured.The kidney governs water. Deficient kidney yang fails to govern qi transformation, leading to water flooding over the skin and edema. "discusses the edema caused by the pathogenesis of kidney deficiency in DKD. DKD has the clinical manifestation of kidney deficiency,and modern physicians have counted the clinical information of the four diagnoses of DKD[14].Among them, fatigue, frequent nocturnal urination, fear of cold in limbs, fear of cold in waist and knees and so on are all clinical manifestations of kidney deficiency.Proteinuria in the course of DKD is closely related to renal deficiency. Rensong Yue[15] considered that protein is a minor important substance in human body, and DKD proteinuria is closely related to the loss of storage function of kidney.Dong Wang[16] believed that the pathogenesis of kidney deficiency runs through the whole process of DKD, and the decrease of storage function caused by kidney deficiency was an important cause of proteinuria, so the medicine of tonifying kidney and astringent essence should be applied to this disease all the time. Wind evil is an important part of the pathogenesis of DKD. As early as in The Yellow Emperor's Inner Classic: Basic Questions recorded the kidney disease caused by wind evil: Basic Questions·The Treatment Acupoint of Water Disease and Fire Disease and Synopsis of the Golden Chamber Shuiqi disease records that A strong person overworked, resulting in kidney deficiency and sweating, just at this time attacked by the wind evil which can not enter the viscera and not go out from the skin,staying in Xuanfu, under the skin, would suffer from edema disease.This discussed the mechanism of the occurrence of nephropathy caused by the external wind evil. Basic Questions·Special Diseases and Basic Questions·Fengxie discussed the clinical manifestations of Shenfeng disease mainly with edema. Contemporary doctors[17]summed up the main points of the diagnosis of wind pathogenic injury to the kidney, including facial swelling, exogenous lung channel symptoms, excessive urine foam, rheumatism symptoms and occurrence in winter and spring, and so on. Zhao Jinxi [18] observed that DKD had common manifestations of wind poison and internal wind, such as thin and itchy skin, pain, dizziness, leg and foot cramps and so on. The nature of wind is Yang evil, and its nature is opposite to the storage function of kidney,which disturbs the kidney and impairs the storage function of the kidney. Proteinuria is an important indication of “wind”[19].Professor Guo Ziguang[20] a great master of traditional Chinese medicine, also proposed that, “if lung deficiency cannot be controlled, kidney deficiency cannot be sealed,and wind pathogen interferes with the kidney, proteinuria can be seen every time”.Pearson correlation analysis of a clinical retrospective study also confirmed that there was a positive correlation between the factor score of wind evil syndrome and 24-hour urine protein in DKD[21].

AO-SR is a representative medicine pair of tonifying kidney and dispelling pathogenic wind. In modern research [21-23],The effective ingredients of AO mainly include sterols, diphenylheptanes,flavonoids, phenols, ethers and fatty acids. It has antioxidant,antiallergic, neuroprotective, anti-inflammatory and other pharmacological effects. The effective ingredients of SR mainly include chromones, vanillin, acidic polysaccharides, volatile oil, etc.,which have antipyretic, anti-inflammatory, labor pain, anti allergic and other pharmacological effects. From the above, it can be seen that AO-SR medicine based on the method of tonifying kidney and dispelling pathogenic wind is effective in the treatment of DKD, but there are still some problems, such as unclear pharmacodynamic material basis and unclear action mechanism, so it is difficult to conduct a comprehensive and systematic study from the whole to the molecular level.Therefore, the network pharmacology is used to predict and analyze its effective ingredients, action targets and pathways in the treatment of DKD.

There are 324 target proteins in the effective components of AOSR drug pair screened through the traditional Chinese medicine data platform. According to the network pharmacology analysis,there are 48 key target genes of DKD and AO-SR. In the study of the components of AO ,Li et al[24]. found that AOE,the extract of Yizhiren, could effectively inhibit the urine volume of rats, while large doses of AOE could increase the excretion of K+. Through Xie et al. [25] further research on AOE, he found that AOE can reduce the blood glucose level and renal pathological damage of mice by regulating the composition of intestinal flora; Yiyao Chen et al.[26]can reduce intracellular fat through anti oxidative stress by treating protocatechuic acid and chrysin in AO extract, which has a protective effect on cells. For the study of the composition of SR,Li Li et al.[27] has proved that the compounds of SR chromogenic ketones have antioxidant effect. Zeqing Zhang et al.[28] through the scavenging effect of SR polysaccharide on diphenylpicryl radical,superoxide anion and hydroxyl radical in vitro, it was proved that SR polysaccharide had strong antioxidant activity, among which acidic SR polysaccharide (A-SPS) had the best effect.Caining Zhang et al. [29] through the study on the extraction of SR volatile oil, it was found that SR volatile oil has certain free radical scavenging ability and antioxidant effect. Meanwhile, Li Wen et al. [30] found that SR has the effect of anticoagulant and reducing plasma concentration in the study of anticoagulant pharmacology of SR.

By gene enrichment prediction, The effect of AO-SR pair on DKD may be mainly through NF kappaB, classical MAP kinase,JNK and p38 MAP kinase, ERK5 and other pathways. Among them, NF kappaB pathway has been proved to be induced by lipopolysaccharide in the experiment of emodin regulating macrophage polarization, which plays a key role in the inflammatory cascade cascade cascade [31]JNK pathway plays an important role in many physiological and pathological processes such as cell proliferation, transformation, differentiation and apoptosis[32].Some scholars use curcumin analogue C66 to selectively inhibit JNK pathway, and it has better effect on DKD in vivo and in vitro[33],and Chen Yufeng et al. [34]found that JNK pathway could block the expression of NLRP3 protein induced by high glucose;Our team has also proved in early studies that AO can play a role in DKD disease by inhibiting the inflammatory pathway of NLRP3 [10].P38 MAP kinase pathway has been proved to be sensitive to stress,hypoxia, inflammation and other stimuli, and can participate in many processes such as inflammation, cell differentiation, apoptosis and so on[35]. Some scholars have found that p38 MAP kinase pathway plays an important mediating role in the inflammatory response.When p38 MAP kinase pathway is inhibited, it can alleviate the DKD process by reducing the inflammatory response and urine protein [36-37]. Wenxia Liu's team [38]found that under the condition of high glucose and high fat, inhibition of ERK5 pathway can alleviate apoptosis and fibrosis induced by high glucose.At the same time, through animal experiments, some scholars found that the time of thrombosis was delayed compared with the blank group after inhibiting ERK5 pathway, which confirmed that ERK5 pathway played an important role in platelet aggregation in vivo[39].

Through the study of network pharmacology, it is found that the AO-SR medicine pair based on the method of tonifying kidney and expelling pathogenic wind is realized through multi-components,multi-targets and multi-pathways in the treatment of DKD, which is the same as the overall concept of TCM and the concept of drug compatibility and efficiency. Most of the existing researches on AOSR medicine pair are based on single ingredient and single machine system, and the effective ingredients, action pathway and mechanism are not perfect. The purpose of this study is to provide more research programs for the treatment of DKD with AO-SR medicine pair based on the method of tonifying kidney and expelling pathogenic wind through the prediction of network pharmacology, and to prove part of the mechanism of AO-SR in the treatment of DKD, so as to provide new ideas and methods for the later research of AO-SR drug pair in the treatment of DKD.