Gynostemma Pentaphyllum Ameliorates Lipid Metabolic Abnormalities in Diabetic Kidney Disease

Background: Patients with diabetic kidney disease (DKD) were often accompanied with dislipidemia. Gynostemma pentaphyllum can ameliorate insulin resistance and reduce the synthesis of triglycerides and cholesterol, but the underlying mechanism is still unclear. Therefore, we used the network pharmacologic strategies to evaluate potential therapeutic effects and protective mechanisms of gynostemma pentaphyllum on diabetic kidney disease. Methods: Gynostemma pentaphyllum's potential targets were predicted using the TCMSP databases. The pathogenic factors involved in DKD and dislipidemia were screened by the OMIM and Gene Cards databases. The common targets of gynostemma pentaphyllum, DKD and dislipidemia were used to establish a protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to explore the potential molecular pathways. Results: The key targets for the therapeutic effects of gynostemma pentaphyllum included IL-6, AKT1, VEGFA, PTGS2, CCL2 and CASP3. Enrichment analysis showed that the underlying mechanism were mainly the involved in inhibition of inammatory response, negative regulation of apoptotic process and angiogenesis. TNF, PI3K-Akt, and HIF-1 signaling pathways were considered as the key pathways. Conclusion: Gynostemma pentaphyllum played a therapeutic role in DKD complicated with dislipidemia, mainly through inuencing inammation response, apoptosis and angiogenesis.


Introduction
Nowadays, with the changing of people's lifestyles, diabetic mellitus has become a global problem that mainly affects developing countries. According to epidemiological statistics, diabetics worldwide is expected to reach 366 million by 2030 [1,2]. Diabetic kidney disease (DKD) is a typical microvascular complication of diabetes and primary cause for end-stage kidney disease, and the number of patients with DKD will exceed 100 million by 2030 [1]. Therefore, an effective strategy for preventing DKD is urgently needed.
In general, patients with DKD often exert a variety of lipoprotein abnormalities [2]. In 1982,Moorhead et al [3] found that hyperlipidemia was associated with glomerular capillary injury, and suspected that the continuous ltration of lipids and lipoproteins promoted the development of kidney damage. Studies on animal models indicated that hyperlipidemia was closely related to lipid deposition in mesangium and the progress of early diabetic kidney lesions [4].
Long-term hyperglycemia leads to higher levels of low-density lipoprotein (LDL) cholesterol and lowers levels of high-density lipoprotein (HDL) cholesterol, resulting in abnormal lipid metabolism [5].
Futhermore,Tolomen et al, [6]demonstrated that a higher high-density lipoprotein cholesterol level is associated with a lower incidence of chronic kidney disease. The primary cause of diabetic dyslipidemia is the increased free fatty-acid (FFA) released by fat cells [7][8][9][10]. Excessive FFAs can be converted to TG by deserti cation, which deposit in renal tissues and cause toxicity, aggravating kidney injury [11], Furthermore, the impaired ability to inhibit FFAs release can enhance the production of very low-density lipoprotein (VLDL) cholesterol in the liver [12]. Dyslipidemia may aggravate renal damage in diabetic patients by affecting the coagulation system and damaging endothelial cells [13].
Hyperlipidemia in diabetic kidney disease is the disease controlled by multiple genes and targets. Drugs with highly selective ligands targeting a single target often cannot change the overall state of the disease and it is di cult to effectively control or prevent the development of the disease. In 2004, Morphy et al provided a new thinking for therapy: multi-target therapeutics [14].
Due to the complexity of multi-component and multi-target for gynostemma pentaphyllum, it is di cult to accurately elaborate the corresponding relationship between components and targets. However, network pharmacology based on system biology provides us with the possibility of systematic study of drug action network and helps to reveal the potential mechanism of gynostemma pentaphyllum ameliorates lipid metabolic abnormalities in DKD.

Materials And Methods
Target screening TCMSP (http://www.tcmspw.com/tcmsp.php) with the keyword of "Gynostemmae Pentaphylli Herba" was employed to collect the active ingredients of gynostemma pentaphyllum. TCM is often clinically used via oral administration. Thus, the active ingredients of gynostemma pentaphyllum were selected based on oral bioavailability (OB) > 30%, drug-likeness > 0.18. The overlapped targets collected from TCMSP and SymMap (https://www.symmap.org/) were used to obtain the potential targets of gynostemma pentaphyllum. Genecard (https://www.genecards.org/) and DisGeNET (https://www.disgenet.org/home/) were used for the related target search with the keywords of "hyperlipidemia", "hypercholesterolemia", "hypertriglyceridemia", "diabetic kidney disease" and "diabetic nephropathy". The common targets from these two databases were taken to obtain the related targets associated with hyperlipidemia and DKD. Then, the potential targets of gynostemma pentaphyllum were mapped onto the related targets of hyperlipidemia and DKD. Venn diagram for mapped targets was made by Venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/).

Network construction
The active ingredients and corresponding targets of gynostemma pentaphyllum were imported into the Cytoscape3.7.2 (https://cytoscape.org/) to make the network platform. A protein-protein interaction (PPI) network was established to elucidate the associations between potential targets and other proteins [30]. The disease targets and common targets were imported into STRING tools (https://string-db.org/), followed by the construction of PPI network models using Cytoscape3.7.2.

Results
To elucidate the mechanisms underlying the inhibition of the abnormal lipid metabolismby gynostemma pentaphyllum in DKD, we performed a series of bioinformatic analyses on several public datasets ( Figure  1). Totally, 202 active ingredients of gynostemma pentaphyllum were obtained by TCMSP, of which 24 reached the criteria of OB > 30% and DL > 0.18 (Table 1). Cyclobuxine exhibited the highest bioavailability (84.48%), indicating that it can be utilized by the human body to a high degree. Gypentonoside A_qt had the highest DL (0.8), suggesting its high probability for being used as a drug.
As shown in Figure 2A, quercetin was the active ingredient correlated with most of the target genes. Two types of gypenosides, gypenoside XXVII_qt and XXVIII_qt, were also connected to some target genes. These results indicated that quercetin and gypenosides might play key roles during the whole process. After overlapping the disease genes derived from Genecard and DisGeNET databases, 289 common genes were obtained, and 267 target interactions were obtained from these overlapped genes. As shown in Figure 2B, INS (insulin), ALB (albumin), IL-6 (interleukin-6), AKT1(AKT Serine/Threonine Kinase 1), TNF (tumor necrosis factor) and VEGFA (vascular endothelial growth factor A) might play key roles in hyperlipidemia in DKD.
Thus, the genes involved in in ammation, apoptosis and angiogenesis, including IL-6, PTGS2, CASP3 and VEGFA, were selected for molecular docking analysis with Rhamnazin and quercetin. As shown in Figure   5, the molecular interaction between VEGFA and quercetin (VEGFA-quercetin) was the closest interaction, indicating its critical role in attenuating the lipid metabolic abnormalities in DKD by gynostemma pentaphyllum.

Discussion
Three signi cant signaling pathways (hsa04066, hsa04668, hsa04151) were selected as the critical pathways by PPI networks and enrichment analysis of common-targets, which were mainly related to three functional modules including in ammation, apoptosis and angiogenesis. Therefore, this research was focused on these three modules.
It was reported that gynostemma pentaphyllum could remarkably suppress the increase of triglyceride, total cholesterol and LDL-cholesterol in serum caused by high-fat diet [31]. Gao et al found that gynostemma pentaphyllum had the function of hypoglycemic and hypolipidemic by the expression of NFE2-related factor 2 signaling in diabetic rats [32].
In some previous studies, lipid accumulation in the kidney was considered to be the key process in DKD [33][34]. And a research reported that quercetin could alleviate lipid accumulation in the kidney of DKD rats by regulating the expression of sterol regulatory element-binding proteins (SREBPs) and LDL receptor protein, which were controlled by Akt [35]. In addition, quercetin could also regulate the expression of insulin receptor substrate and glucokinase and improved the insulin secretion function of β-cells [36]. In terms of blood lipid, a study showed that 1% quercetin diet could reduce the content of FFA in serum of rats and promoted the catabolism of fat [37].
Gypenosides could signi cantly reduce the insulin resistance parameters and increased the glycogen concentration [38]. As well, gypenosides could effectively prevent hyperlipidemia and atherosclerosis. Some related researches found that theregulation of blood lipid was related to the inhibition of FFA produced by adipocytes and the promotion of neutral fat synthesis [39][40]. Therefore, quercitrin and gypenosides may be key ingredients in the therapy. The in ammatory model showed that gynostemma pentaphyllum may in uence in ammation through HIF-1 signaling pathway and TNF signaling pathway. Pro-in ammatory cytokines IL-6 was the key gene in these two pathways. Senn et al found that IL-6 could inhibit the insulin signal transduction and insulin action [41]. And another study reported that the release of IL-6 could weaken the function of pancreatic β cells, leading to insulin resistance, insulin secretion dysfunction and the occurrence of metabolic syndrome [42]. Therefore, abnormal secretion of IL-6 may result in the hyperlipidemia in DKD by affecting insulin. In addition, a signi cant inhibition of IL-6 was observed in the gypenosides concentration of 150 μg/ml and 200 μg/ml [43]. It was reasonable to believe that gynostemma pentaphyllum may inhibit the expression of IL-6, which furthermore would relieve the DKD. Some in ammatory cytokines, such as TNF-α, could activate the NF-κB signaling pathway, which would aggravate the in ammatory response and accelerate the development of DKD [44]. And a signi cant inhibition of TNF-α was observed in the gypenosides concentration range of 100-200 μg/ml [43]. These results indicated that gynostemma pentaphyllum may inhibit the expression of TNF-α to improve in ammation and lipid metabolism in DKD.
HIF-1 signaling pathway and PI3K-Akt signaling pathway acted as the crucial roles in apoptosis and angiogenesis. And HIF-1α as a transcription factor could stimulate the expression of cytokines related to renal interstitial cell brosis, which would aggravate the diabetic kidney injury [45][46]. Some animal studies indicated that HIF could cause the increase of insulin sensitivity as well as the decrease of serum cholesterol levels [47,48]. The mechanism might be associated with the reduction of metabolite accumulation in glycolysis and TCA cycle in diabetic renal cortical tissue [49]. These results showed that HIF stabilization could ameliorate lipid metabolic abnormalities in DKD. Another study reported that quercetin could scavenge the ROS to reduce the level of HIF-1αand reduce the level of caspase-9 in H 2 O 2treated cells, suggesting that quercetin could inhibit the caspase-dependent apoptosis [50]. These results showed that gynostemma pentaphyllum could inhibit the cell apoptosis as a HIF stabilizer, which may be related to the mechanism that gynostemma pentaphyllum ameliorated the lipid metabolic abnormalities in DKD.
PI3K and the downstream effector Akt belonged to the signal transduction enzymes and was involved in regulating cellular activation and apoptosis [51]. Li et al [52] found that the PI3K signaling pathway played a crucial part in autophagy and DKD, of which the mechanism may be related to the podocyte adhesion injury. In terms of tumors, Maurya et al reported that quercetin modulated the PI3K-AKT signaling pathway and reduced the Akt and PDK1 phosphorylation [53], which was similar to the mechanism that gynostemma pentaphyllum ameliorated lipid metabolic abnormalities in DKD.
VEGF was a growth factor for endothelial cell, which played a crucial part in the process of angiogenesis [54]. VEGFA was closely related to angiogenesis, which was mediated by VEGF receptor 1 (VEGFR1) and VEGFR2 signaling pathways [55][56]. A study found that quercetin can signi cantly suppress the activation of VEGFR2 downstream molecules, to inhibit angiogenesis, such as AKT and mTOR [57]. Few studies have reported that gynostemma pentaphyllum improved the symptoms of DKD through increasing the angiogenesis, more research is needed to con rm these points in the future.

Conclusion
In summary, gynostemma pentaphyllum could ameliorate the abnormal lipid metabolism in DKD through in uencing in ammation response, apoptosis and angiogenesis. However, the research on speci c cell or gene expression is not in-depth enough, which will be further explored in follow-up experiments. Figure 1 A owchart for researching therapeutic mechanism of gynostemma pentaphyllum on hyperlipidemia in DKD.