Gentiopicroside Ameliorates Diabetic Renal Tubulointerstitial Fibrosis via Inhibiting the AT1R/CK2/NF-κB Pathway

Renal tubulointerstitial fibrosis (TIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, is the typical pathological alteration in diabetic nephropathy. Gentiopicroside (GPS), a natural compound with anti-inflammatory activity, has been demonstrated to alleviate glomerulosclerosis, whereas whether GPS inhibits TIF via regulating inflammation remains unclear. In this study, diabetic db/db mice and high glucose (HG)-stimulated renal tubular epithelial cells (NRK-52E) were applied to explore the effects and mechanisms of GPS on TIF. The results in vivo showed that GPS effectively improves glycolipid metabolism disorder, renal dysfunction, and TIF. In particular, GPS treatment reversed the abnormal expressions of EMT marker proteins including elevated α-smooth muscle actin and vimentin and decreased E-cadherin in the kidney of db/db mice. Moreover, GPS treatment also inhibited protein expressions of angiotensinⅡ type 1 receptor (AT1R) and CK2α and the activation of the NF-κB pathway. Importantly, the aforementioned effects of GPS acted in vivo were further observed in vitro in HG-stimulated NRK-52E cells, which were independent of its effects on glucose and lipid-lowering activity but were reversed by AT1R over-expression. Together, our results indicate that GPS that directly inhibits the CK2/NF-κB inflammatory signaling pathway via AT1R may also contribute to the amelioration of TIF in diabetes.


The effects of GPS on Vimentin and α-SMA in db/db mice were detected by immunohistochemistry
The results of immunohistochemistry were quantitatively analyzed by the software of Image J. The data showed that GPS treatment could inhibit the high expressions of α-SMA and Vimentin in db/db mice (Supplement Figures S1A,B).

Mechanism of GPS for DN treatment was predicted based on network pharmacology
The method of network pharmacology was adopted to predict the mechanism of GPS for DN treatment according to reported method (Xiong et al., 2020). Firstly, the chemical structure and 3D file of the GPS were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Then potential targets of GPS were obtained from the Swiss Target Prediction (http: //www.swisstargetprediction.ch). After that, some target manes need to be normalized by Uniprot (https://www.uniprot.org/) and Homo sapiens were kept for further analysis. The search keyword "diabetic nephropathy" was used in Gene Cards database (https://www.genecards.org/) and DN related genes were obtained. Next, the DN-related genes were compared with the potential targets of GPS, providing potential targets of GPS for anti-DN. Later, above targets were entered into String database (https: //string-db.org/) with the species being limited to Homo sapiens, and the Protein-Protein Interaction (PPI) network with a score greater than 0.7 was retained. The PPI was obtained and the results of the Kyoto Encyclopedia of Genesand Genomes (KEGG) pathway annotation were used to analyze the PPI network and the key proteins were selected for further analysis.From above analysis, we known that intervening RAS was the main mechanism of GPS for DN treatment (Supplement Table 1).

HG increased migration ability of NRK-52E cells
The effect of HG on the migration ability of NRK-52E cells was detected by cell scratch test, and scratch areas were quantitatively analyzed by the software of Image J (Supplement Figure S1C).

MTT assay
MTT assay showed that GPS with the concentration less than 800 μM had no cytotoxicity to NRK-52E cells (Supplement Figure S1D).

GPS inhibited HG-induced migration of NRK-52E cells
The effect of GPS on the migration ability of HG-induced NRK-52E cells was detected by cell scratch test, and scratch areas were quantitatively analyzed by the software of Image J (Supplement Figure S1E).

The distributions of p65 in nucleus and cytoplasm under the HG conditions at various times were detected by western blot
After stimulation with HG for 30 min, the p65 expression in nucleus was significantly increased (Supplement Figure S1F).

Plasmid transient transfection
Compared with normal group, AT1R expression significantly increased accompanied by a striking increase in GFP expression after the NRKA-52E cells were transfected with over expression plasmid targeting AT1R (Supplement Figure S2A).

AT1R over-expression can reverse the inhibitory effect of GPS on NRK-52E cells migration
The scratch areas were quantified by the software of Image J. The data showed that the migration ability of NRK-52E cells incubated with HG was increased obviously and the decreased migration was observed after cells were treated with GPS. However, the increased migration was detected again after cells were treated with GPS and over expression plasmid of AT1R (Supplement Figure S2B).

AT1R over-expression can reverse the inhibitory effect of GPS on p65 distributed in nucleus
As shown in immunofluorescence, the phenomenon of p65 translocation into nucleus was inhibited after GPS intervention. Nevertheless, AT1R over-expression up-regulated the expression of p65 in nucleus (Supplement Figure S2C).

The co-localization of CK2α and IκBα in cytoplasm
As shown in immunofluorescence, the co-localization of CK2α and IκBα in cytoplasm was observed in NRK-52E cells (Supplement Figure S2D).