WEI Ting-ting, MENG Li-feng, LI Li-rong, ZHANG Peng, HUANG Xue-xia

1.Guangxi University of traditional Chinese Medicine, Nanning 530000, China

2.The First Affiliated Hospital of Guangxi University of traditional Chinese Medicine, Nanning 530000, China

Keywords:

ABSTRACT Diabetic nephropathy (DN) is the most serious microvascular complication of diabetes mellitus,which is highly prevalent worldwide.Abnormal activation of Wnt/β -catenin signaling pathway is an important mechanism of renal damage induced by hyperglycemia.Many studies have shown that TCM has the advantages of high efficiency and safety in the prevention and treatment of DN.Some TCM monomers and compounds repair podocyte function and inhibit transdifferentiation process by inhibiting the activation of Wnt/β-catenin signaling pathway,thus playing a protective role in kidney.Based on this, this paper will review the existing research results and related mechanisms of TCM intervention in Wnt/β-catenin signaling pathway in the treatment of DN, in order to promote the more effective and reasonable application of TCM in clinical practice.

Diabetic Nephropathy (DN) is a serious glomerular complication caused by Diabetes Mellitus (DM).According to the International Diabetes Federation (IDF), about 40% of diabetic patients can develop advanced renal failure[1].As of 2017, there were about 114 million DM patients in China, and the incidence of diabetes was 10.9%.However, a meta-analysis by Zhang[2] estimated that the prevalence of DN in China was as high as 21.8%.DN is the most common cause of Chronic kidney disease (CKD) in developed countries, and the largest cause of end-stage renal disease (ESRD)in China[3-4].DM and CKD are independent risk factors of all-cause death and cardiovascular accidents.Compared with DM and CKD,DN has the highest risk of death[5].With the development of DN,renal structure changes, including deposition of extracellular matrix(mainly mesangium), thickening and proliferation of glomerular basement membrane, which ultimately lead to glomerular sclerosis and renal interstitial fibrosis[6].Currently, there is no precise therapeutic target or drug for DN in Western medicine, and ACEI/ARB antihypertensive drugs with the effect of reducing urinary protein and kidney protection are mainly used as first-line drugs, but the effectiveness and safety of these drugs are controversial due to adverse reactions such as elevated serum potassium and creatinine[7].Studies have shown that the activation of Wnt/β-catenin pathway is involved in the occurrence and development of DN through the regulation of glucolipid metabolism, inflammatory response,podocellular repair and epithelial mesenchymal transition (EMT)[8,9].In recent years, some scholars have shown that Traditional Chinese medicines such as Huayu Tongluo[10]and Yishen Huayu Prescription[11] can regulate Wnt/β-catenin signaling pathway,inhibit mesenchymal transformation and repair podocellular damage, thus improving the prognosis of DN.In this paper,the mechanism of Wnt/β-catenin signaling pathway in DN is systematically summarized, and the TCM compounds targeted to this signaling pathway for DN treatment are summarized as follows.

1.Overview of Wnt/β-catenin signaling pathway

The Wnt protein family is composed of 19 secreted glycoproteins of Wnt protein members, which are mainly divided into Wnt1 and Wnt 5A, which activate typical Wnt pathways (Wnt/β-catenin) and atypical pathways (Wnt/PCP and Wnt/Ca2+), respectively[12].Wnt/β-catenin signaling pathway is highly conserved during embryonic development and is involved in regulating various biological processes such as cell differentiation, proliferation, adhesion and apoptosis, adult stem cell renewal and tissue homeostasis[13].When the Wnt/β-catenin pathway is inactivated, the tumor suppressor Axin (Axin) and adenomatous colonic polyposis (APC) form a so-called destruction complex containing glycosylated synthase kinase 3β (GSK3β), which phosphorylates β catenin with casein kinase 1(CK1).It has been labeled as ubiquitination and proteasome degradation[14,15].In contrast, when the Wnt ligand binds to the Frizzled protein (Frizzled,Fz)/ low-density lipoprotein-associated receptor protein (LRP5/6) complex, inhibiting the activity that destroys the complex, β-catenin in the cytoplasm stabilizes and accumulates, After reaching a certain concentration, translocation to the nucleus binds with transcription factor T cytokine 4 (TCF4)/lymphocyte enhancer factor 1 (LEF1) to recruit different nuclear regulatory factors, thereby activating the expression of downstream genes related to the Wnt pathway and generating various inflammatory mediators and fibrosis factors to induce disease[16,17].Activation of this pathway is associated with a variety of chronic kidney disease, cancer, osteoporosis, and degenerative diseases[18].

2.Relationship between Wnt/β-catenin signaling pathway and diabetic nephropathy

The main types of DN podiocytes injury include disappearance of foot processes, dedifferentiation, mesenchymal transformation and apoptosis, which can lead to damage of the structure and function of glomerular filtration barrier.Under the condition of high glucose,podocytes induce the upregulation of vascular endothelial growth factor (VEGF), and the excessive production of mesangial cell matrix leads to glomerulosclerosis, highlighting the importance of podocytosis in the pathogenesis of DN[19-20].Previous studies have found that high expression of Wnt proteins (Wnt1, Wnt4) and β-catenin can be observed in podocytes of DN animal models, and the expression of Nephrin is inhibited and podocytes are damaged,confirming the correlation between abnormal activation of Wnt/β-catenin and the occurrence of DN[21].Stimulated by various factors such as high glucose and inflammatory factors, abnormal activation of Wnt/β-catenin signaling pathway mediates multiple target genes involved in the occurrence of EMT[22].The formation of EMT is characterized by loss of e-cadherin protein function,decreased expression of podocin, and new mesenchymal cell phenotypes such as α -smooth muscle actin (α-SMA), increased expression of desmin, type ⅰ collagen.Is an important link in the formation of renal fibrosis[23,24].Increased expressions of Wnt4,β-catenin and α-SMA were observed in the renal tissue of diabetic nephropathy rat model, and e-cadherin protein decreased, resulting in renal tubulointerstitial EMT, which further demonstrated that Wnt/β-catenin pathway plays a pivotal role in the process of DN renal fibrosis[25].Therefore, the intervention of Wnt/β-catenin pathway is expected to be a breakthrough in the treatment of diabetic nephropathy and has clinical practical significance.

3.Intervention of Traditional Chinese medicine on Wnt/β-catenin signaling pathway in the treatment of diabetic nephropathy

DN belongs to the category of diseases and syndromes such as“Xiaodan”, “edema” and “urine turbidity” in traditional Chinese medicine.It is one of the common symptoms in the middle and late stages of diabetes..According to traditional Chinese medicine, the pathogenesis of DN is always characterized by the deficiency of origin, while the deficiency and the fact are mixed with each other.In the process, the pathological products such as phlegm turbidity and blood stasis block the kidney collateral, aggravate the disease, and form a vicious cycle that causes the disease to be prolonged and not cured[26,27].In addition, laboratory tests show that the hemodynamic abnormalities and hypercoagulability of DN patients are similar to the “blood stasis” syndrome of TCM[28].Based on this, TCM can treat DN by intervening Wnt/β-catenin signaling pathway,regulating and alleviating podoccyte injury, improving glucose and lipid metabolism, inhibiting EMT and other mechanisms, which has clinical practical significance[29,30].

3.1 Regulation of Wnt/β-catenin signaling pathway in diabetic nephropathy by Monomers and extracts of Traditional Chinese medicine

Salvia miltiorrhiza is a classic Chinese medicine for promoting blood circulation and removing blood stasis.Its main active ingredients are phenolic acids and diterpenoid quinones.Huang B Y et al[31] found that tanshinone IIA can increase the expression of e-cadherin while decreasing the expression of α-SMA, and reduce the ectopic expression of β-catenin, and prevent EMT in human renal tubular epithelial cells HK-2 induced by high glucose.Xiang et al[32] used salvia miltiorrhiza extract to intervene in DN model rats and found that salvia miltiorrhiza extract significantly reduced the high expression of WNT-4, β-catenin and TGF-β in the renal tissues of rats, and improved the symptoms of renal tubular epithelial cell vacuolation, glucolid metabolism disorder, inflammatory cell infiltration and so on.Previous reports have also indicated the possibility of interaction between Wnt4/β-catenin pathway and TGF-β1 pathway in DN patients[30].Therefore, it is concluded that Salvia miltiorrhiza can regulate Wnt/β-catenin and TGF-β related signaling pathways to inhibit transdifferentiation, improve renal interstitial fibrosis, and achieve the purpose of TREATING DN.Panax notoginseng has the function of removing blood stasis and filling deficiency.Xie[33]used Sanqi granules to treat DN rats by gavage, and the results showed that compared with the model group,protein contents of Desmin, α-SMA, Wnt1 and β-catenin in Sanqi group were significantly reduced, while the content of transmembrane protein Nephrin was increased.These results indicated that Panax notoginseng inhibited the Wnt/β-catenin signaling pathway in rat kidney tissue and improved the podocellular transdifferentiation induced by high glucose, thus delaying the progression of DN.Astragalus membranaceus, as a classic qi tonic, is often used in the treatment of chronic kidney disease.Bao Fang et al[34] used APS to intervene in the renal tubular epithelial cell injury model induced by high glucose, and found that APS reversed the trend of axin-1 decrease and β-catenin increase by inhibiting the inactivation of Wnt signaling pathway, thus protecting epithelial cells.Wang et al[35]confirmed that astragaloside IV can inhibit oxidative stress injury and inflammatory response mediated by high glucose, and prevent EMT progression by regulating Wnt/β-catenin signaling pathway.Tripterygium wilfordii has anti-inflammatory, immunosuppression,repair podocyte and other modern pharmacological effects[36].Shi G et al[24] found that the protein levels of Nephrin and Podocin in mouse podocyte in low-dose triptolide group were restored, and the protein levels of type I collagen, snail factor, Wnt3α and β-catenin were down-regulated.It is speculated that the mechanism may be related to the inhibitory effect of triptolide on Wnt3α/β-catenin signaling pathway.Chang B C [37] proposed that Tripterygium wilfordii polyglycosides inhibited the excessive activation of Wnt/β-catenin and NF-κB pathways in diabetic rats, and reduced the protein contents of WNT-1, P-GSK-3 β, β-catenin and TGFβ1, alleviating inflammatory stimulation and improving renal pathological damage.Cornus officinalis has the functions of tonifying liver and kidney, astringent fixation and stopping band.Chen Ye et al[38] found that Cornus officinallis can reduce the mRNA and protein expressions of Wnt4, β-catenin and YKL-40 in rat kidney tissue, revealing that the therapeutic mechanism of Cornus officinallis on DN is related to the inhibition of YKL-40 and Wnt/β-catenin signaling pathway.Rhubaric acid has anti-inflammatory,antioxidant stress and anti-fibrosis effects, and exists in a variety of Traditional Chinese medicines[39].Duan et al[40] reported that rhubaric acid can down-regulate Wnt1, P-GSK-3 β, and P-β-catenin levels in renal tissues of diabetic rats, while simultaneously increasing nephrin expression and protecting podocyte from injury.Zhang T [41] also confirmed that rhubarb can down-regulate the expression of Wnt, β-catenin protein and TGF-β1, and inhibit the proliferation of glomerular mesangial cells induced by high glucose.It is concluded that the regulation of Wnt/β-catenin pathway is one of the mechanisms of rhubaric acid in the treatment of diabetic nephropathy.

3.2 Regulation of Wnt/β-catenin signaling pathway in diabetic nephropathy by Chinese herbal formula

Song W G et al[11] found in animal experiments that Yishen Huayu Prescription could inhibit the activity of Wnt/β-catenin pathway,increase the expression of Nephrin and e-cadherin protein in kidney tissues of DN rats, decrease the expression of Wnt1, β-catenin and α-SMA, repair podocyte injury in rats, and improve related indicators.Zhang Lin[42] explored the renal protective effect of Shenshuaining capsule on DN, and the results showed that the expression levels of WNT-1 and β-catenin (P ＜ 0.05) and the levels of urinary protein, SCr and urea nitrogen (BUN) were significantly decreased in shenshuaining group.It was confirmed that Shenshuaining capsule can effectively regulate Wnt/β-catenin pathway to play an effect on DN.Bai Lu et al.[10] used the selfdesigned Stasmostasis and collateral-removing TCM granules to intervene in DN rats, and also observed that the activity of Wnt/β-catenin pathway in kidney tissue of rats in the TCM group was down-regulated, and the expression levels of PRO, Wnt4 and β-catenin in 24 h urine were significantly reduced.CAI Jingzhao et al[43] found that Tangshen Ping could increase nitric oxide (NO)and superoxide dismutase (SOD) while reduce the expression levels of propylene glycol (MDA), Wnt4, β-catenin and other proteins,and reverse the occurrence process of EMT in DN KKAy mice.The mechanism may be related to the regulation of Tangshenping on Wnt/β-catenin signaling pathway and anti-oxidative stress.Yang Na et al[44] conducted a diabetic rat model experiment, and the results showed that the expression of Wnt4 and β-catenin and FBG decreased in the kidney tissue of the jiangtang Prescription group, indicating that Shiyiyishengjiangtang Prescription could inhibit the high expression of Wnt/β-catenin signaling pathway,increase insulin sensitivity and improve insulin resistance.Zou et al[45] found that renal function, urinary protein and lipid metabolism disorders of RATS were improved after shenan granules were used to treat DN rat models, and the expressions of Wnt pathway-related proteins Wnt1 and β-catenin were down-regulated.Liu et al[46]applied Liuwei Dihuang Pill to treat DNOP rat model, and the data showed that Liuwei Dihuang pill not only inhibited the expression of α-SMA, Wnt1, β-catenin and histone demethylase (KDM7), but also decreased the levels of TNF-α, IL-6, IL-8 and IL-1β.It also increased serum calcium levels.These results indicate that Luwei Dihuang Pill can inhibit inflammatory response and treat DN related osteoporosis by down-regulating KDM7A and Wnt/β-catenin signaling pathways.Qishenyiqi dropping pill is a traditional Chinese medicine compound preparation made by extracting the active ingredients of Astragalus membranaceus, salvia miltiorrhiza, Panax notoginseng and incense lowering, which is mainly used in clinical adjuvant treatment of myocardial infarction and heart failure[47].Zhang et al[48] applied it in the treatment of DN in an innovative way, and observed that Qishenyiqi dripping pill down-regulated the expressions of Wnt1, β-catenin, Smad2, TGF-β, type I collagen,α-SMA and FN in diabetic rats, and improved the indicators of SCr, BUN and 24h Pro.This study proved for the first time that Qishenyiqi dripping pill can play a protective role in kidney by inhibiting Wnt/β-catenin and TGF-β/Smad2 signaling pathways.In conclusion, TCM can delay renal interstitial fibrosis and improve renal function by regulating the Wnt/β-catenin signaling pathway,inhibiting the epithelial-mesenchymal transformation process,reducing inflammation, regulating glucose and lipid metabolism and other aspects, revealing the advantages of TCM monomers and compounds with multiple components, multiple targets, multiple pathways and multiple effects.

4.Summary

Through the intervention of traditional Chinese medicine in Wnt in recent years/ β- After summarizing the relevant literature research on the treatment of DN in the catenin signaling pathway, it can be found that most of the Chinese herbal medicine targeting the pathway to prevent and treat DN has the functions of Invigorating Qi,nourishing kidney, activating blood circulation, dredging collaterals and removing blood stasis.Its good clinical curative effect confirms many doctors’ understanding of the etiology and pathogenesis characteristics of diabetic nephropathy, such as Qi deficiency and blood stasis, kidney deficiency and blood stasis[49].In addition, after studying the relevant experiments of traditional Chinese medicine compound intervention in this pathway, it can be seen that most of its prescriptions follow the treatment principle of “attacking and tonifying both” and often take strengthening the spleen and kidney,dredging the Fu organs and removing turbidity, promoting blood circulation and removing blood stasis as the treatment methods,which are also quite effective.

Relying on the development of current emerging technologies,the pharmacological research of single components of traditional Chinese medicine and the efficacy experiment of traditional Chinese medicine compound continue to deepen, and traditional Chinese medicine is used to intervene Wnt/ β- Catenin signaling pathway has shown great potential in the treatment of diabetic nephropathy.However, according to the current research status, Wnt/ β- The research of catenin signaling pathway mostly focuses on Wnt1 and Wnt4 proteins, and the research of other family members is relatively few.Based on the complexity of traditional Chinese medicine components, we should further explore the role of traditional Chinese medicine in Wnt from the aspects of molecular,cytology and genetics in the future/ β- The specific mechanism of catenin signaling pathway in the treatment of DN.In terms of longterm efficacy and drug toxicology, more comprehensive and in-depth experimental research is needed to raise the current clinical evidence to the evidence-based level in order to better serve the clinic.

Author’s Contribution

This paper is written by Wei Tingting; Huang Xuexia provided comments and suggestions on the research direction of the review;Li Lirong searched relevant literature; Zhang Peng participated in proofreading the document format and details; Meng Lifeng participated in the correlation review and article polishing of literature review and research direction.