Identification of the Main Chemical constituents and mechanism of Renshen Guben oral liquid against Renal Fibrosis

High-throughput identification of chemical constituents in RSGB using UPLC-QTOF-MS/MS

In order to describe the chemical fingerprint of RSGB, UPLC-QTOF-MS/MS was used for sample collection and UNIFI 1.8 software for identification [12, 13]. The base peak intensity (BPI) chromatograms of RSGB in positive and negative ion modes were shown in Fig. 1A and B. A total of 201 compounds were identified or tentatively characterized in both the positive (109) and negative (125) ion modes under optimized conditions, including Triterpenes (49), Phenols (46), Other Terpenoids (28), Iridoids (19), Saccharides (16), Ketones, Aldehydes, Acids (16), Phenylpropanoids (9), Organic acids (6), Flavonoids (5), Steroids (4), Sterols (1), Amino acids (1) and Others (27) (Fig. 1C), 41 of which were identified in Ginseng radix et rhizoma, 41 in Rehmanniae radix, 34 in Moutan cortex, 28 in Corni fructus, 20 in Alismatis rhizoma, 20 in Rehmanniae radix praeparata, 16 in Poria, 15 in Asparagi radix, 9 in Dioscoreae rhizome and 6 in Ophiopogonis radix (Fig. 1D). The detailed information of the 201 compounds was listed in Table S2, S3, containing Component name, Observed neutral mass (Da), Observed m/z, Formula, Mass error (ppm), Observed RT (min), Adducts, MS/MS, Category and Herbs.

Fig. 1figure 1

RSGB chemical profile analysis. (A and B) BPI chromatograms of positive ion mode (A) and negative ion mode (B); (C and D) Classification of constituents in RSGB according to chemical structures (C) and herbs (D). RS is Ginseng radix et rhizoma, DH is Rehmanniae radix, SDH is Rehmanniae radix praeparata, SY is Dioscoreae rhizoma, SZY is Corni fructus, MDP is Moutan cortex, ZX is Alismatis rhizoma, FL is Poria, MD is Ophiopogonis radix, TD is Asparagi radix

To further improve the accuracy of the identification, 15 standards were used for comparative analysis, including ginsenoside Rg1, ginsenoside Rg2, ginsenoside Rc, ginsenoside Ra1, ginsenoside Rb1, ginsenoside Re, rehmannioside A, rehmannioside D, loganin, loganic acid, gallic acid, cornuside, paeonol, apiopaeonoside and paeonolide. Altogether 9 compounds in positive ion mode and 13 compounds in negative ion mode corresponded to the standards (Additional file 1: Fig. S1A-1D), thus proving the accuracy of the component identification RSGB.

Ginsenoside Rg1, rehmannioside D, paeonolide and gallic acid are typical components of Ginseng radix et rhizoma, Rehmanniae radix, Moutan cortex and Corni fructus, respectively, and their fragmentation patterns were analyzed as examples. ⑴ Ginsenoside Rg1 is a triterpene with an accurate molecular weight of 800.4910 (C42H72O14). In the negative ion mode, its quasi-molecular ions were m/z 799.4863 [M-H]− and m/z 845.4891[M + HCOO]−. As the glycosidic bond breaks, 162-fragments are gradually lost, forming [M-H-Glc]− (C36H62O9, m/z = 637.4314) and [M-H-2Glc]− (C30H52O4, m/z = 475.3778) (Fig. 2A). This fragmentation pattern is also applicable to ginsenoside Rb1, Rc, Re, Ra1, etc., so that other triterpenoid saponin can be accurately identified. ⑵ Rehmannioside D is an iridoids with an accurate molecular weight of 686.2260 (C27H42O20). In the negative ion mode, its quasi-molecular ions were [M-H]− (m/z = 685.2159), and [M + HCOO]− (m/z = 731.2248). The glycosidic bond of [M-H]− breaks to form [M-H-Glc]− (C21H30O14, m/z = 505.1523). Then lost a C6H11O5 to form [M-H-2Glc- C6H11O5]− (C15H18O9, m/z = 341.1057), and lost a C9H8O3 to form [M-H-2Glc-C6H11O5-C9H8O3]− (C6H12O6, m/z = 179.0561), or shed a H2O to form [M-H-2Glc-C6H11O5-H2O]− (C15H16O8, m/z = 323.0973) (Fig. 2B). According to this pattern, iridoids such as rehmannioside A, catalpol and gardoside could also be identified. ⑶ Paeonolide is a glycoside with an accurate molecular weight of 460.1586 (C20H28O12). In the positive ion mode, paeonolide lost a C9H10O3 to form [M + H-C9H10O3]+ (C11H18O9, m/z = 195.1027), meanwhile, it also can lost a C11H18O9 to form [M + H-C11H18O9]+ (C9H10O3, m/z = 162.0724) (Fig. 2C). Similarly, apiopaeonoside also can be identified. ⑷ Gallic acid is a polyphenol with an accurate molecular weight of 170.1200 (C7H6O5). In the negative ion mode, gallic acid lost a molecule of COOH to form [M-H-COOH]− (C6H6O3, m/z = 125.0238), or lost a H2O to form [M-H-H2O]− (C7H6O5, m/z = 169.0143) (Fig. 2D). Similarly, protocatechuic acid, 4-hydroxybenzoic acid, methyl gallate, etc. can be accurately identified.

Fig. 2figure 2

Mass spectrometry of ginsenoside Rg1, rehmannioside D, paeonolide and gallic acid standards and their fragmentation patterns. A Ginsenoside Rg1, B Rehmannioside D, C Paeonolide and D gallic acid

The protective effect of RSGB on mice UUO model

Ligation of the left ureter is the most common method in UUO model and has been widely adopted to establish animal models of interstitial fibrosis [15]. To investigate the influence of RSGB on renal fibrosis, C57BL/6J mice were treated with RSGB for 14 days after surgery. The results showed that serum Scr and BUN levels of UUO model were significantly increased compared with that of Sham (Fig. 3A and B), which was consistent with the obvious injury in the kidney tissue we have observed. After RSGB treatment, Scr and BUN levels have been significantly suppressed (Fig. 3A and B). HE staining and Masson staining showed that the renal tissue structure of mice in the sham group was normal, no obvious inflammatory cell infiltration was observed in the renal interstitium, and only a small amount of bright blue collagen fibers was observed in the interstitium and around the blood vessels. In the model group, a large number of inflammatory cells were infiltrated into the renal tissue, vacuolar lesions were found in the renal tubules, part of lumen was dilated, the epithelial cells of the renal tubules were atrophied, and there was edema in the renal interstitium, with a large number of blue collagen fibers. However, the above lesions were significantly improved by RSGB and benazepril, the arrangement of tissue cells tended to be orderly, and the bright blue collagen fibers were significantly reduced (Fig. 3C-E). These results indicated that RSGB alleviates renal fibrosis in mice UUO model.

Fig. 3figure 3

Beneficial effect of RSGB on UUO mice. A and B The levels of BUN, and Scr in serum detected by biochemical kits; C A statistical diagram of fibrosis rate in each group. D and E HE and Masson staining of renal tissues. **P < 0.01 and *P < 0.05 vs. Sham, ##P < 0.01 and #P < 0.05 vs. UUO.

RNA sequencing analysis of RSGB on renal fibrosis

To elucidate the mechanism by which RSGB alleviate renal fibrosis, RNA sequencing was performed (n = 4). As shown in the volcano map and heat map, some mRNA levels in UUO mice altered significantly after RSGB treatment (Fig. 4A-D). Differential gene (FC > 1.5 and P < 0.05) screening showed that, a total of 587 genes were regulated by RSGB, among which 226 genes (92 up-regulated and 134 down-regulated) were regulated by RSGB to a level close to that of the Sham group, which were called “direct effector genes” (Fig. 4E). The 226 direct effector genes were subjected to GO analysis, as shown in Fig. 4F, the biological process (BP) included positive regulation of kidney development, regulation of kidney development, and nephron development, indicating the regulation of RSGB on kidney. At the same time, pathway analysis of 226 effector genes was carried out based on KEGG database. The top ten pathways regulated by RSGB include PPAR signaling pathway, Focal adhesion, Wnt signaling pathway and MAPK signaling pathway (Fig. 4G). Importantly, these pathways have been reported to be regulated in kidney disease.

Fig. 4figure 4

RNA sequencing analysis of RSGB on UUO mice. A and B Volcano diagram between Sham vs. UUO (A) and UUO vs. RSGB (B); (C and D) Heat map between Sham vs. UUO (C) and UUO vs. RSGB (D); (E) Heat map of 226 effect genes in UUO mice after RSGB treatment. (F) GO biological process analysis of 226 effect genes of RSGB; (G) KEGG pathway analysis of 226 effect genes

Multi-dimensional network analysis of RSGB curing renal fibrosis

For further elucidate the mechanism of RSGB, Qi-Yin deficiency syndrome (QYDS) (the main syndrome of RSGB treatment) was jointly analyzed. The relationship between UUO model and QYDS was explored by SoFDA (https://cn.string-db.org/). The 743 differential genes (FC > 10) of UUO/Sham were input into SoFDA for TCM syndrome enrichment, and the results showed that they were involved in Syndrome of Yin Depletion (P = 0.00277), Syndrome of Yin Deficiency (P = 0.012), and Qi-Yin Deficiency Syndrome (P = 0.0178) (Fig. 5A), indicating that the UUO model we established conformed to the characteristics of QYDS. Therefore, the combined analysis of disease, syndrome and formula genes were carried out. The genes of three groups were subjected to KEGG pathway analysis, and the pathways with P < 0.05 were selected for intersection analysis. As shown in the Venn diagram, disease, syndrome and formula genes had a high correlation (Fig. 5B).

Fig. 5figure 5

Gene analysis of disease, syndrome and formula. A The syndrome enrichment analysis of UUO/Sham differential genes was conducted by SoFDA. B KEGG pathway analysis of disease, syndrome and formula genes was carried out by DAVID, and pathways of P < 0.05 were analyzed by Venn diagram

PPI network analysis of disease, syndrome and formula genes was performed by STRING, and core genes were obtained by screening Degree, Closeness and Betweenness values, and KEGG pathway was enriched by DAVID. Finally, 23 pathways with the highest relevance to UUO model were retained. By analyzing the genes involved in the 23 pathways and RSGB constituents related to these genes, the “constituents-targets-pathways” network was constructed, including 26 key constituents of RSGB and 88 corresponding key genes, which were subdivided 26 disease genes, 27 syndrome genes, and 48 formula genes (Fig. 6). The pathways could be divided into 4 functional modules according to their pharmacological effects: ⑴ Immune and anti-inflammatory related pathways, including NF-kappa B signaling pathway, MAPK signaling pathway, TNF signaling pathway, TGF-beta signaling pathway, AMPK signaling pathway, Th17 cell differentiation, PI3K-Akt signaling pathway, Wnt signaling pathway, mTOR signaling pathway, Toll-like receptor signaling pathway, JAK-STAT signaling pathway and T cell receptor signaling pathway; ⑵ Metabolism related pathway, including Calcium signaling pathway, Sphingolipid signaling pathway, cAMP signaling pathway and Lipid and atherosclerosis; ⑶ Cell junction pathway, including Gap junction, Focal adhesion, Adherens junction; ⑷ Other signaling pathway, including Apoptosis, Cell cycle, Rap1 signaling pathway, Ras signaling pathway.

Fig. 6figure 6

Multi-dimensional network of “Constituents - Targets - Pathways”. Red diamonds represent constituents, ellipses represent targets, and blue diamonds represent pathways. Targets are classified according to their attribution to disease, syndrome, or formula

Among the 26 key constitutes, 8 of them were derived from Ginseng radix et rhizoma (ginsenoside Rg1, ginsenoside Rg2, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Ra1, ginsenoside Rc, ginsenoside Re, ginsenoside F1), 6 from Rehmanniae radix / Rehmanniae radix praeparata (raffinose, leonuride, catalpol, melittoside, rehmannioside D, rehmaionoside A), 5 from Moutan cortex (galloyl oxypaeoniflorin, paeonoside, paeonol, apiopaeonoside, paeonolide), 5 from Corni fructus (cornuside, loganin, loganic acid, gallic acid, methyl gallate), 2 from Poria (poricoic acid G, pachymic acid), 1 from Alismatis rhizoma (raffinose). Some key components of RSGB have been reported to act on anti-inflammatory and renal protection. Ginsenoside (Rg1, Rg2, Rg3, etc.) [16,17,18], paeonol [19, 20], gallic acid [21], loganin [22] and pachymic acid [23] showed activities in kidney fibrosis and other kidney diseases. Cornuside [24], loganic acid [25] and rehmannioside A [26] have anti-inflammatory activities, which further proved that the key constituents in the “constituents-targets-pathways” network may be used as the pharmacodynamic material basis of RSGB in the treatment of renal fibrosis.

Mechanism verification of RSGB alleviating renal fibrosis

As shown in the “constituents-targets-pathways” multi-dimensional network, RSGB mainly regulates the immune inflammation pathway. Combining differential genes in the RNA sequencing, we selected nuclear factor- kappa B 1 (NFKB1), NFKB2, TGFB1, WNT4, nerve growth factor receptor (NGFR) involved in immune inflammation and cell cycle to perform quantitative real-time PCR (qRT-PCR). The results demonstrated that RSGB significantly altered these gene abnormalities induced by the UUO model (Fig. 7A). In order to further verify the effects of RSGB on TGFβ-1 signaling pathway, Wnt signaling pathway and NF-κB signaling pathway, the expression levels of Tgfβ1, Smad2/3, Wnt4, β-Catenin, NGFR, NF-κB p65, phospho-NF-kB p65, phospho-NF-kB p50 and DDIT3 in Sham, UUO and RSGB groups were determined by WB experiment. In UUO model group, Tgfβ1/Smad2/3 pathway, Wnt4/β-Catenin pathway, and NGFR/NF-κB pathway were activated, which led to inflammatory response and accelerated cell proliferation, thus promoting the progression of renal fibrosis. After treatment with RSGB, the levels of these proteins were significantly reduced (P < 0.05), indicating that RSGB inhibited the activation of these pathways (Fig. 7B). Therefore, our study demonstrated that RSGB suppressed the inflammatory response and cell cycle by inhibiting the Tgfβ1/Smad2/3 pathway, Wnt4/β-Catenin pathway and NGFR/NF-κB pathway, thereby exerting an anti-renal fibrosis effect (Fig. 7C).

Fig. 7figure 7

The mechanism of RSGB on UUO model. A The genes relative expression levels were detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001. B The levels of Tgfβ1, Smad2/3, Wnt4, β-Catenin, NGFR, NF-κB p65, phospho-NF-kB p65, phospho-NF-kB p50 and DDIT3 in Sham, UUO and RSGB groups were determined by WB. The histograms show the statistics of protein levels (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05. C The mechanism of RSGB against renal fibrosis

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