Yu-hui Ma,Li-na Zhang,Qian Cui,Lin-feng Li,Guo-wei Zhang*

1 College of Chinese Medicine,Hebei University,Baoding,Hebei,China

Background

Diabetic nephropathy (DN), also known as diabetic glomerulosclerosis, is one of the complications of diabetes mellitus and the primary cause of end-stage renal disease and chronic kidney disease. According to the relevant data, the prevalence of DN in Chinese diabetic patients is about 20% - 30%, and the prevalence is increasing year by year[1], which seriously endangers the lives and health of our people.Diabetes mellitus is called"consumptive thirst"in traditional Chinese medicine.And diabetic nephropathy is one of the main chronic complications and an important cause of end-stage nephropathy. Once it develops to end-stage nephropathy,fibrosis will occur in the kidney, which seriously threatens the life and health of patients. Nowadays,western medicine treatment isn’t pretty effective. There are a great number of clinical cases which show that the treatment of traditional Chinese medicine in DN has a remarkable curative effect for decreasing urine protein[2].It can also activates protein kinase C by activating the end products of glycation and polyol pathway to the improve the DN glycometabolic disorder[3].The treatment based on syndrome differentiation with suitable Chinese crude drug can relieve edema[4]thereby improving the clinical symptoms of DN patients and alleviating pain.Through consulting the literature,we found that the study of DN in traditional Chinese medicine mainly focused on etiology, pathogenesis, treatment and summary of prescriptions and medicines,and lacked in in-depth study of the law of drug use and its mechanism of action.Therefore, this paper collects and collates the clinical research literature of Chinese medicine in treating DN in recent 10 years, and analyses its medication rule and action signaling pathway,which can provide reference for clinical research of Chinese medicine in treating DN in the future.

Materials and methods

The sources of medical prescription

All of the literatures are selected from China National Knowledge Infrastructure (CNKI). Click “Advance search”,type the words“DN”or“consumptive thirst with kidney disease” and “Traditional Chinese Medicine” or“Traditional Chinese Medicine Treatment” in the theme.The published time is from January 1,2008 to October 31,2018,and there are 1569 literature in total.

Data screening

Inclusion criteriaAll of the clinical Prescriptions about the treatment of DN by traditional Chinese medicine are involved; there are definite drug composition and dosage in the literature;the prescriptions concerned have definite curative effect; the Chinese medicine formulation is oral decoction or pill.

Exclusion criteriaCases included in the study were complicated with other serious diseases except DN;treated by non-traditional Chinese medicine decoction;only prescription name without drug composition, or incomplete composition; the repeated prescriptions in literature or related researches; invalid or aggravated;unclear prescription dosage identification.

Prescription Screening and the Specification of Drug NameAccording to the standards above all,there are 118 prescriptions and 227 Chinese crude drugs involved.Then according to the “Chinese Pharmacopoeia” of the 2015 edition and Thirteenth Five-Year Plan Tenth Edition of Science Textbook "Pharmacy", the names of the Chinese crude drug are standardized one by one.

Checking and entering

There is one person put the finished prescriptions into TCM Inheritance Assistance Platform (TCMMISS, V2.5)[5], and only the original prescriptions are chosen, the adding and subtracting prescriptions are removed. After the input is completed, two people alternately review the data to ensure the accuracy of the input data.

Prescriptions analysis

Prescription analysis:The frequency and regularity of 118 prescriptions were analyzed by "data analysis" of Traditional Chinese Medicine Inheritance Assistant Platform.Target analysis:The core drug pairs of analysis results were input into BATMAN-TCM [6] online analysis system, and the signal pathways of core drug pairs were analyzed.

Result

The frequency analysis of Chinese crude drug

According to the "frequency statistics" of 227 Chinese medicine in 118 prescriptions,17 of them were used more than 20 times,among which the first seven were Huangqi(Astragalus membranaceus (Fisch.) Bunge), Danshen(Salvia miltiorrhiza Bunge), Fuling (Poria cocos (Schw.)Wolf), Shuyu (Dioscorea opposita), Danggui (Angelica sinensis(Oliv.)Diels),Dihuang(Rehmannia glutinosa(Gaetn.) Libosch. ex Fisch. et Mey), Shanzhuyu (Cornus officinalis Sieb.et Zucc.).(Table 1).

Table 1 Drug use frequency≥20 times

Analysis of Drug Formulation Rules Based on Association Rules

According to the setting of support degree 18 and confidence degree 0.09, through the "medication mode",we analyzed the prescription formulation rules,we got 27 groups of medicine which were commonly used,involving 13 Chinese medicine. According to the frequency of medicine, the top three medicine groups were Shanzhuyu-Fuling, Shanzhuyu-Shuyu,Danshen-Fuling (Table 2). Then the association rule analysis shows that the probability of compatibility of Shanzhuyu, Fuling and Shuyu are 96% in the prescriptions collected for clinical treatment of DN(Table 3). Through the "network display" module, the network display diagram of commonly used drug combinations is drawn(Figure 1).

Table 2 Commonly used drug combinations for treating DN

6 Astragalus membranaceus(Fisch.)Bunge,Dioscorea opposita 27 20 Ophiopogon japonicus(Linn.f.)Ker-Gawl,Schisandra chinensis 19 7 Cornus officinalis Sieb.et Zucc,Dioscorea opposita 26 21 Atractylodes macrocephala Koidz,Poria cocos(Schw.)Wolf 19 8 Salvia miltiorrhiza Bunge,Dioscorea opposita 26 22 Cornus officinalis Sieb.et Zucc,Astragalus membranaceus(Fisch.)Bunge,Dioscorea opposita 19 9 Salvia miltiorrhiza Bunge,Angelica sinensis(Oliv.)Diels 26 23 Rehmannia glutinosa(Gaert.)Libosch.ex Fisch.et Mey,Dioscorea opposita 18 10 Poria cocos(Schw.)Wolf,Alisma plantago-aquatica Linn.25 24 Rehmannia glutinosa(Gaetn.)Libosch.ex Fisch.et Mey,Poria cocos(Schw.)Wolf 18 11 Cornus officinalis Sieb.et Zucc,Dioscorea opposita,Poria cocos(Schw.)Wolf 25 25 Paeonia suffruticosa Andrews,Poria cocos(Schw.)Wolf 18 12 Rehmannia glutinosa(Gaetn.)Libosch.ex Fisch.et Mey,Astragalus membranaceus(Fisch.)Bunge.23 26 Cornus officinalis Sieb.et Zucc,Salvia miltiorrhiza Bunge,Dioscorea opposita 18 13 Cornus officinalis Sieb.et Zucc,Astragalus membranaceus(Fisch.)Bunge 22 27 Astragalus membranaceus(Fisch.)Bunge,Dioscorea opposita,Poria cocos(Schw.)Wolf 18 14 Astragalus membranaceus(Fisch.)Bunge,Angelica sinensis(Oliv.)Diels 22

Table 3 Association rule of drug combinations for treating DN

Figure 1 Commonly used drug combinations network

The potential mechanism analysis of the compatibility of CORNI FRUCTUS, PORIA and DIOSCOREAE RHIZOMA in the treatment of DN based on BATMAN-TCM

The diseases analysis of CORNI FRUCTUS, PORIA and DIOSCOREAE RHIZOMAWe set "Score cutoff"to 80 and "Adjusted P-value" to 0.01,and there are 57 kinds of diseases involving Shanzhuyu,Fuling and Shuyu single drug and the compatibility enrichments (Table 4).We found that the DN-related diseases contain non-insulin-dependent diabetes mellitus,nephropathy and renal failure.

The signal pathway analysis of CORNI FRUCTUS,PORIA and DIOSCOREAE RHIZOMA and the compatibility enrichmentAccording to the results coming from BATMAN-TCM, 16 KEGG pathways are included(Table 5). The pathways relating to DN are VEGF signaling pathway 、 MAPK signaling pathwayandPI3K-Akt signaling pathway and so on.Visualization of potential targets, signaling pathways and disease networks is followed as Figure 2.

Table 4 Analysis results of Shanzhuyu,Fuling and Shuyu single drug and compatibility for diseaseenrichment

13 Cardiovascular disease,unspecified 7 1 3 14 Heart failure 4 2 4 15 Drug dependence 4 1 5 16 erectile dysfunction 7 1 2 17 Arrhythmia 4 1 4 18 prostatic cancer 4 3 2 19 Brain damage 4 2 3 20 Cognitive deficits 4 2 3 21 Respiratory diseases 1 4 4 22 Non-insulin dependent diabetes mellitus 3 2 4 23 Mental illness 1 3 3 24 Cancer,nonspecific 4 2 1 25 ischemic heart disease 2 2 2 26 Chronic ileitis 1 2 2 27 Excessive drinking 2 1 2 28 Nausea and vomiting 3 1 1 29 Inflammatory bowel disease 1 2 2 30 Neurological diseases 1 2 2 31 Pain,nonspecific 3 1 1 32 Alcohol dependence 3 1 1 33 benign prostatic hyperplasia 2 1 1 34 Vomit 1 2 1 35 Paranoia 1 1 1 36 Multiple sclerosis 1 1 1 37 Insulin resistance (obesity related)1 1 1 38 Hypoxic-ischemic encephalopathy 1 1 1 39 Dependence on opioids 1 1 1 40 Ischemia-reperfusion injury 1 1 1 41 Autoimmune diseases 1 1 1 42 Attention deficit hyperactivity disorder 1 1 1 43 Coronary heart disease 1 1 1 44 Gastric emptying disorder 1 1 1 45 Oxygen-induced retinopathy 1 1 1 46 Vitamin D deficiency 1 1 1 47 Dyskinesia 1 1 1 48 Raynaud syndrome 1 1 1 49 Behcet's disease 1 1 1 50 Hypertrophic vascular disease 1 1 1 51 Chaos in Gilles de la Tourette 52 progressive autonomic failure 1 1 1 1 1 1 53 kidney failure 1 1 1 54 nephropathy 1 1 1

Table 5 Analysis result of KEGG pathway

Figure 2 Visualization of potential targets,signaling pathways and disease networks

Discussion

The Pathological Mechanism of DN is complex, it is generally believed at home and abroad that the pathogenesis is related to lipid metabolism,hemodynamic changes,hormone levels and genetic factors and so on[7].Western treatment isn’t pretty effective and has serious side effect. The research of traditional Chinese medicine treatment to DN has come into a deep level, and the clinical effect is good and has a little side effect. Lv Jingxia et al. [8] introduced Professor Liu Wenfeng's understanding of the etiology and pathogenesis of diabetic nephropathy and the self-summarized prescription of Baxian Baoshen Decoction,and illustrated the good effect of Baxian Baoshen Decoction in clinical treatment through real medical records. Zhang Jianghua et al.[9]used in vitro cell culture technology to study the therapeutic effect of traditional Chinese medicine on DN,which concludes that Yiqi Yangyin Xiaozheng Tongluo Recipe has the effect of delaying the development of DN.Dai Xiaoxia et al. made a comparative experiment of traditional Chinese medicine and Western medicine in treating DN[10],which concluded that the curative effect of traditional Chinese medicine is better than that of Western medicine treatment group and proves that traditional Chinese medicine treatment can significantly improve the clinical symptoms of DN. However, the types of DN can’t be used flexibly for its complicate and masses, which is lack of systematic research on evidence-based medicine and pharmacological analysis[11]. Traditional Chinese Medicine Inheritance Assistant Platform System (TCM Inheritance Assistant Platform) is an organic combination of computer science and TCM. It intelligently processes information, follows the concept of evidence-based inheritance ， which realizes the functions of sorting out and analyzing disease information, syndrome information, TCM information,prescription information, medical records and related information [12-13].Network pharmacology is based on system biology to analyze multi-target networks of drugs,regulate signaling pathways, and study the mechanism of drug action to improve curative effect[14-15].Traditional Chinese Medicine Inheritance assistant system combined with BATMAN-TCM database helps to explore the law of traditional Chinese medicine prescription medication,analyze drug targets and disease pathways, which is conducive to modern clinical research of traditional Chinese medicine.

Results: After"Frequency Statistics"we got 17 Chinese crude drugs whose use frequency is more than 20 times among 227 Chinese crude drugs in 118 prescriptions,and among that the first seven is Huangqi, Danshen, Fuling,Shuyu, Danggui, Dihuang, Shanzhuyu. According to the frequency of medicine, the top three medicine groups were Shuyu-Fuling, Shanzhuyu-Shuyu, Danshen-Fuling.Shanzhuyu has the function of tonifying and replenishing liver and kidney,inducing astringency,and can be used to treat internal heat and consumptive thirst. Indian bread has the functions of diuresis and dampness, invigorating the spleen and soothing the mind. It can be used to treat edema and hypouria in clinic. Shuyu has the effect of tonifying qi,nourishing yin,invigorating spleen,lung and kidney.Modern pharmacological studies have shown that Shanzhuyu can reduce blood sugar, diuresis, inhibit platelet aggregation and anti-thrombosis [16-17]; Fuling contains components such as beta-Poriacocosan and Poria acid,which have diuretic,sedative and anti-tumor effects.Poriacocos polysaccharide also has the effect of enhancing immunity and effectively inhibiting renal interstitial fibrosis [18-19]; The yam polysaccharide of Shuyu has the effect of lowering blood sugar [20], can improve the non-specific immune function, specific immune cells and humoral immune function. The chemical constituents of Shanzhuyu, Fuling and Shuyu have certain therapeutic effects on DN. In addition,through the analysis of network pharmacology software,the number of pathways between the compatibility of the drugs and the related disease is 16,which include 6 signal transduction pathways related to DN. By analyzing the traditional experience of TCM in treating DN,we hope to provide ideas and directions for the researches of modern TCM in treating DN.

Through the system of TCM inheritance assistant platform, we get the high-frequency drug compatibility which is Shanzhuyu, Fuling and Shuyu for DN treatment according to association rules. We analyze the compatibility with BATMAN-TCM and excavate its pharmacological mechanism for DN treatment. The results show that they have a tight connection with DN for there are a great number of targets involved in diabetes mellitus and diabetic nephropathy. Nowadays,the pathological mechanism of DN is still not completely clear, but it is generally believed that proteinuria is of great significance in the process of diagnosis and prognosis of diabetic nephropathy [21]. The study finds that the targets of the drug compatibility have a close connection with the pathways of proteinuria like VEGF and TGF-β and so on.In some researches,Shanzhuyu can inhibit oxidative stress molecules in kidney,reduce blood sugar level, reduce proteinuria, slow renal failure and improve clinical symptoms [22-23]; Poriacocos polysaccharide can protect renal function and delay renal failure in chronic kidney disease, Zhang Huiru et al.found that as the increase of the dosage of Poria Cocos polysaccharide[24], 24-hour proteinuria content in diabetic nephropathy rats increased which had statistical significance, and the experiment confirmed that Poriacocos polysaccharide was beneficial to reduce proteinuria, increase plasma protein content and protect renal function. What’s more, Huangling et al. had an animal experiments and proved that yam polysaccharide,the active ingredient of Shuyu [25], could reduce the levels of urinary protein, urea nitrogen and creatinine caused by diabetic nephropathy, which was beneficial to prevent and reduce renal injury. In conclusion, the combination of Shanzhuyu, Fuling and Shuyu is beneficial to reduce proteinuria, alleviate renal fibrosis and improve clinical symptoms of DN.

In this paper, traditional Chinese medicine (TCM)inheritance assistant platform and network pharmacology research methods were used to analyze the rule of TCM in treating diabetic nephropathy and related drug signal transduction pathways. It was concluded that Shanzhuyu,Fuling and Shuyu had certain positive effects in preventing and treating diabetic nephropathy, and could be used as compatibility of traditional Chinese medicine in treating diabetic nephropathy, and a new way of thinking to clinical treatment is provided.

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