Active ingredients in P. vulgaris, target molecules and enriched biological processes relevant to analgesic and glucose intolerance
To understand the relationship between anti-inflammation and metabolic dysregulation during PE treatment, common genes of analgesic and glucose intolerance were extracted from the database in Genecards. A total of 2954 in glucose intolerance-related module and 1069 in analgesic-related module were yield respectively, among which 547 genes were their common genes (data not shown). That is to say, 51.2% analgesic-related genes are associated with the occurrence and/or development of glucose intolerance. However, only 8 out of 547 genes appeared as validated P. vulgaris target genes in TCMSP database, namely GCG, CCK, PYY, RHO, THBD, INS, TYR, MPO (Fig. 1A). The range of their relevance scores for glucose intolerance was 79.3(INS)-3.4(RHO), and 5.1(CCK)-0.44(THBD) for analgesic. These target genes were associated with only 6 out of the 60 compounds in P. vulgaris, including oleic acid, rutin, quercetin, palmitic acid, p-coumaric acid and luteolin (Fig. 1A). Herein, oleanolic acid and its derivatives may have therapeutic effects on different diseases and symptoms, including inflammation . Stigmasterol, a steroid alcohol, is found in numerous medicinal plants, vegetables, and nuts, was reported to alleviate cutaneous allergic responses in animal models .
Beyond the above 8 target molecules, the molecule with highest relevance score for glucose intolerance was glucose-6-phosphatase catalytic subunit 1(G6PC1) (89.1), a rate-limit enzyme for hydrolyzing glucose 6-phosphate resulting in the elevation of plasma glucose level. G6PC1 is primarily expressed in the liver and the kidney, and is up-regulated by glucocorticoids through the induction of the binding with glucocorticoid receptor (GR, or NR3C1) to its upstream glucocorticoid responsive element (GRE) (-231 to -129). GR is one of the 547 target genes with relevance score as 9.4 for glucose intolerance, and 0.6 for analgesic. GR may be activated by Δ7-stigmasterol and oleanolic acid in P. vulgaris suggesting as a possible compound-target molecule bridge for the downstream signaling [15, 17]. GILZ, SGK and GSK are GC-responsive genes; meanwhile, they are also in the module of analgesic-glucose intolerance. Their relevant scores are 2.04, 3.4, and 1.3 for analgesic, 13.4, 37.7 and 13.6 for glucose intolerance respectively, indicating their close relationship with anti-inflammation and glucose regulation. In addition, another very important regulatory molecule, BDNF, is also one of the common genes, with relevance score of 2.6 for analgesic, and 9.3 for glucose intolerance, indicating it may play an important role for anti-inflammation and glucose disposal. Some important cytokines like IL1β, IL6 and IL10, but not limit to, are common genes too. The above genes are also P. vulgaris compound targeting molecules.
By using Cytoscape (version 11.0), a construction of compound–target-disease networks was built (Fig. 1A). Their connections among all involved proteins was visualized using String (version 11.0) (Fig. 1B), and GO enrichment analysis revealed that INS, AKT1, IL6, GCG, NPY, BDNF, GCR, AGRP, CCK and PYY were hub genes within the network (Fig. 1C). These molecules actively involved into 9 biological processes, sc. neuroactive ligand-receptor interaction, PI3K-Akt signaling pathway, FoxO signaling pathway, cAMP signaling pathway, regulation of lipolysis in adipocytes, adipocytokine signaling pathway, insulin secretion, AGE-RAGE signaling pathway in diabetic complications, insulin resistance signaling pathways. Functional analysis using KEGG revealed 13 enriched signaling pathways, shown in Fig. 1D (p < 0.05, q < 0.05). The above evidence indicates that GCR, BDNF etc. intensively involve in the concurrence of analgesic and regulation of glucose homeostasis.
Analgesic effect of PE treatment and its regulation on plasma cytokines
Peripheral analgesic effect of PE was evaluated through acetic acid-induced writhing test, and expressed as inhibition of writhing frequency and percentage relative to control group. Both aqueous PE35 and 70 significantly reduced the number of writhes compared to control group (p < 0.05, Fig. 2A). Their inhibitory percentages were 28.5% and 55.8% respectively (Fig. 2B). The application of mifepritone (2.5 mg/kg/day) diminished PE-initiated analgesic effect to the control group level. Dexamethasone significantly decreased the analgesic response compared with control group (p < 0.05). The decrease was about the same amplitude as that of PE70 treatment (Fig. 2A, B).
Central analgesic effect was evaluated using hot plate test. After treatment for 20 days, PE35, PE70 and Dex treated groups had significant increased latencies to lick the hind paw from 18.9 ± 1.46 (control group) to 23.2 ± 1.86, 27.3 ± 1.96 and 28.4 ± 1.1s respectively (p < 0.05, n = 8). The increase was diminished by the application of mifepristone (p > 0.05 vs control group), suggesting the central analgesic effect is glucocorticoids relevant (Fig. 2C).
Rotarod test revealed that both PE35 and PE70 treatments significantly reduced the period the animals remained on the rod after a 20-day treatment compared to the control group (p < 0.05). Mifepristone was able to rescue the decrease (p > 0.05 vs control) (Fig. 2D), suggesting that PE may have a systemic effect on the animals, and the effect of PE on neuromuscular coordination is through GC signaling.
Plasma IL1β, IL6 and IL10 were tested 3 weeks after treatment to ascertain PE dependency of the inhibition of pro-inflammatory cytokines. IL1βwas inhibited 20.2% and 34.1% by PE35 and PE70 respectively (Fig. 2E) (p < 0.05). Consistently, IL6 was 222.3 ± 18.8 (9.6%) and 195.4 ± 14.1pg/ml (20.7%) in PE35 and PE70 groups respectively, and they were significantly lower than that of control group, 246.4 ± 21.6pg/ml (Fig. 2F) (p < 0.05). Similarly, IL10 level was down-regulated to 81.9 ± 9.1 (13.2%) and 68.7 ± 6.5pg/ml (27.1%) compared to 94.2 ± 12.6pg/ml in control group (Fig. 2G) (p < 0.05). RU486 significantly relief the PE inhibitory effect for the above cytokines (p > 0.05 vs control group) (Fig. 2E, F, G). The above results are consistent with the previous work on the regulatory effect of GCs on plasma cytokines .
PE inhibitory effects on body weight gain, food intake and appetite
To determine whether PE treatment altered metabolism, mouse body weight was tracked during the experiment. Body weight gain was significant decreased two weeks after PE treatment compared to the control mice. Body weight of PE70 group increased 3.16 ± 0.13 g, whereas the control group increased 4.51 ± 0.31 g (n = 8, p˂005). After a 4-week treatment, their body weight comparison became 5.0 ± 0.33 vs 6.34 ± 0.39g (n = 8, p˂005). Dexamethasone treatment demonstrated a similar trend as PE35 treatment (data not shown). The relative body weight change was plotted as Fig. 3A. Meanwhile, PE70 treated mice appeared less food intake after 2 week treatment compared to control mice (n = 8, p˂0.05) (Fig. 3B). PE35 treatment had the same trend but with less amplitude compared to PE70 for food intake and body weight (Fig. 3A, B), indicating the decrease is PE dose dependent. Mifepristone treatment recovered both food intake and body weight gain to the same level as the control group (Fig. 3A and 3B).
To further test the PE effect on appetite control, after the 20-day PE treatment as previous described, mice were fasted overnight. Then their food intake was measured 0.5h, 1h, 4h after food was returned. PE35 and PE70 treatments significantly reduced food intake compared with control mice (Fig. 3C), suggesting PE treatment decreases appetite. Correspondently, PE70 and PE35 treated-mice gained less weight (p < 0.05, Fig. 3D). However, the ratio of body weight gain to the amount of food intake, which is an indicator of calories deposit as body weight [25, 26], was significantly higher in PE70 group from 0.5h to 4h (Fig. 3E), suggesting PE treatment actually increases the tendency of body weight gain relative to food consumption. This evidence gives an interpretation that short-term glucocorticoid therapy does not result in increased body weight, which occurs during long-term therapy .
To detect the underlying mechanism associated with body weight and appetite, gene expression analysis was performed using real time PCR. Two hypothalamic orexigenic molecules, NPY (Fig. 3F) and AGRP (Fig. 3G), were highly decreased to 0.21 ± 0.09 and 0.006 ± 0.285 fold by PE70 compared to control mice (P < 0.05), which is consistent to the low appetite. Mifepristone recovered PE inhibitory effect, suggesting the central effect of PE treatment is through GC signaling. Dexamethasone treatment also gave similar effects as PE70 (data not shown). Hypothalamic BDNF analysis revealed that PE35 and PE70 treatments significantly increased BDNF level to 3.45 ± 0.27 and 5.70 ± 0.53 folds respectively compared to control group (Fig. 3H) (P < 0.05), which is in line with the down-regulation of hypothalamic NPY and AGRP in current study, and appetite increased by BDNF knockdown shown in our previous work .
Impairment of glucose tolerance by PE treatment
To test the effect of PE treatment on glucose metabolism, after the 20-day PE treatment, mice were fasted overnight, and basal blood glucose level was measured followed by IP injection of glucose (1.5g/kg). Plasma glucose was measured every 15min afterwards. Both PE35 and PE70 treatments led to a lower fasting plasma glucose level (p < 0.05, n = 8) (Fig. 4A). To demonstrate PE effects on glucose disposal, glucose level was normalized to that of starting point of each mouse. Relative levels to the starting point were plotted as in Fig. 4B, showing blood glucose level in PE35 and PE70 groups surged significantly higher than control group 15 min after glucose injection, and maintained until 30 to 45 minutes afterwards (p < 0.05, n = 8) (Fig. 4B). The AUCs of PE35 and PE70 group were increased by 115% and 135% respectively compared with control group. The AUC of mifepristone treated group was about identical to the control group (99.5%) (Fig. 4C).
To investigate the underlying mechanism of the over-abrupt of plasma glucose level in PE treated mice, hepatic glucose-6-phosphatase catalytic-subunit-encoding gene (G6PC1) expression in the liver was checked using real time PCR. G6PC1 mRNA level was increased by 1.83 ± 0.27 and 2.78 ± 0.31 folds in PE35 and PE70 groups respectively compared with control mice (p < 0.05, n = 4) (Fig. 4D). Mifepristone inhibited the PE-induced G6PC1 mRNA level (p > 0.05 vs control). The above evidence indicates that PE treatment induces gluconeogenesis in the liver by upregulating G6Pase expression, which leads to the impairment of glucose tolerance. Hepatic BDNF was down-regulated by PE35 and PE70 to 0.70 ± 0.11 and 0.42 ± 0.07 folds respectively (p < 0.05) (Fig. 4E).
Elevated plasma GC and BDNF levels and the expression of hepatic GC-target molecules by PE treatment
Measurement of plasma glucocorticoids revealed that GC level in control group was 2.94 ± 0.47ng/ml. PE35 and PE70 treatments significantly increased GCs to 9.1 ± 1.8 and 15.7 ± 2.3ng/ml (Fig. 5A) (p < 0.05). Meanwhile, plasma BDNF levels were significantly elevated to 143.7 ± 13.1 and 176.6 ± 18.4 pg/mol by PE35 and PE70 respectively compared to 118.4 ± 13.7 pg/mol in control group (n = 8) (p < 0.05, n = 8). PE70 increased significantly higher than PE35 treatment (p < 0.05) (Fig. 5B). Mifepristone administration dramatically diminished PE70 effect to control group level (Fig. 5B, p < 0.05).The elevated level of plasma BDNF was consistent with its expression in the hypothalamus (Fig. 3H), and other organs (data not shown), but not the liver (Fig. 4E). The corresponding increase of plasma BDNF and GC level indicates their close interplay and the possible mechanism of the concurrence of analgesic and glucose intolerance when PE is chronically applied.
To further confirm the GC-like effects of PE treatment, the transcriptional expression of GC-target genes, including glucocorticoid-induced leucine zipper (GILZ), serum-and glucocorticoid-induced protein kinase 1(SGK1), Glycogen synthase kinase 3 beta (GSK3 β) and serine/threonine-specific protein kinase (Akt2/PKB), was quantified by real time PCR. Hepatic GILZ, which serves as a mediator of the anti-inflammatory effects of glucocorticoid in a variety of cells , was increased by 2.98 ± 0.41 folds in PE70 group (p < 0.05) (Fig. 5C). Hepatic SGK1, which accelerates the development of metabolic syndrome including glucose intolerance , was highly increased by 15.55 ± 1.43 folds (p < 0.05) in PE70 group (p < 0.05) (Fig. 5D). Hepatic GSK3 β was mildly but significantly increased 1.4 ± 0.11 (p = 0.0484) and 1.78 ± 0.26 (p = 0.007) folds by PE35 and PE70 respectively (Fig. 5E). GSK3β was originally found to inactivate glycogen synthase and mediate the development of insulin resistance, then identified as a potential target of the therapy for diseases associated with inflammation . In contrast, Akt2/PKB, another GC-target molecule inhibiting glucose release from hepatocytes and increasing insulin sensitivity , was also highly up-regulated by 21.41 ± 2.48 folds in PE70 group (Fig. 5F). The above evidence suggests that PE treatment intensifies GC down-stream signaling in the liver closely relevant to anti-inflammation and glucose intolerance.