Effects of Gancao Nourish Yin Decoration on Liver Metabolic Proles in hTNF-α Transgenic Arthritic Mouse Model


 BackgroundGancao Nurish Yin (GCNY) decoction has been applied to clinical rheumatoid arthritis (RA) patients and it had shown effectiveness not only in disease activity controlling but also on improving patients’ physical status. However, its mechanism of function has not been investigated. Metabolic perturbations have been associated with RA and targeting the metabolic profile is one of the ways to manage the disease. The aim of this study was to observe the effect of GCNY on metabolic changes of human tumor necrosis factor alpha (hTNF-α) transgenic arthritic mouse model. MethodshTNF-α transgenic arthritic model mice were divided into control and GCNY groups with 6 mice in each group. After 8 weeks of treatment, liver tissues of mice in both groups were obtained for liquid chromatography-mass spectrometry (LC/MS) analysis. Significantly regulated metabolites by GCNY treatment were first identified, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and network analysis.ResultsOf the 126 metabolites detected in the liver, 17 metabolites were with significantly altered between control group mice and mice treated with GCNY. Specifically, thiamine, gamma-L-Glutamyl-L-valine, pantothenic acid, pyridoxal (Vitamin B6), succinic acid, uridine 5'-diphospho-glucuronic acid (UDP-D-Glucuronate), uridine, allantoic acid, N-Acetyl-D-glucosamine, nicotinamide ribotide and N2,N2-Dimethylguanosine were down-regulated by GCNY treatment whereas isobutyrylglycine, N-Acetylcadaverine, N-Carbamoyl-L-aspartic acid, L-Anserine, creatinine and cis-4-Hydroxy-D-proline were up-regulated. Six metabolic pathways were significantly altered including the alanine, aspartate and glutamate metabolism, pyrimidine metabolism, thiamine metabolism, amino sugar and nucleotide sugar metabolism, pantothenate and CoA biosynthesis and citrate cycle (TCA cycle). Integrative metabolic network analysis suggested the possibility of GCNY having both positive and negative effects on RA through the suppression of angiogenesis and promotion of leukocyte extravasation into the synovium, respectively. ConclusionsGCNY can induce a change in the metabolic profiles of hTNF-α transgenic arthritic mouse model. Further optimization of this decoction may lead to better therapeutic effects on RA patients.

still lacks in terms of symptom relief and mending joint structural damage [2]. RA in Traditional Chinese Medicine (TCM) is known as Bi -the physical pain or numbness caused by wind, cold and humidity which can cause the stagnation of Qi and blood stasis [3]. Multiple TCM treatment methods including acupuncture, moxibustion and herbal medicine have been applied as complementary management of RA and many effective bene ts have been reported [3]. Among the plethora of herbal medicine available, Gancao Nurish Yin (GCNY) can be an effective complementary medicine for RA with our clinical work showing dramatic bene ts for RA patients. GNCY is a herbal formula developed from The Golden Chamber which is a classical medical book authored by Zhang Zhongjin during the Han dynasty (AD. 150 -154). The formula was associated to acute or chronic gastrointestinal in ammation and Huhuo disease (similar to Behcet's syndrome, an autoimmune disorder) [6]. Although its constituents such as gancao (glycyrrhizae) [7], ginseng [8], ginger [9] and others were reported to be anti-in ammatory by an abundance of research, little is known about the integrative mechanism of the formula which will be more of signi cance from a clinical perspective.
Many evidences suggest that cellular metabolic alterations fuel and dictate the in ammatory state of cells. Data from RA patients demonstrated a strong link between the degree of systemic in ammation and the development of insulin resistance [10]. The induction of arthritis in mice resulted in a global in ammatory state that is characterized by defective carbohydrate and lipid metabolism in different tissues [11]. Therapeutic strategies based on tighter control of in ammation provide promising approaches to normalize and prevent metabolic alterations associated with RA [12].
For animal models of RA, human tumor necrosis factor alpha (hTNF-α) mimics human RA clinical presentations including polyarticular swelling, impairment of movement, synovial hyperplasia and cartilage and bone erosion [13]. The systematical and joints manifestations are stable and has been applied in metabolic researches [14]. Thus, in this study, we investigate the effects of GCNY decoction on the metabolic pro les of hTNF-α transgenic arthritic mouse model.

Animals and grouping
Human TNF-α transgenic arthritic model mice were bought from Guangdong Experimental Animal Monitoring Institute, China. The transgenic mouse line was produced using a human TNF/β-globin (TNFglobin) recombinant gene construct which contained a 2.8kb fragment of entire coding region and promoter of the hTNFα gene fused to a 0.77kb fragment of 3′ untranslated region (UTR) and polyadenylation site of human β-globin replacing that of the hTNFα gene. The fragment was then microinjected into the pronuclei of FVB/J inbred strain fertilized eggs which were subsequently implanted into the fallopian tube of 8-week-old female pseudo-pregnant ICR mice. Transgenic lineages were established by back-crossing the transgenic founder individuals to the FVB/J inbred strain [13].
The hTNF-α transgenic arthritic model mice at 16 weeks of age (all male, 28-33g) were divided into control and GCNY groups with 6 mice in each group. The GCNY group was treated with GCNY decoration (provided by Qianzheng Health Technology Development Co., Ltd. Shanghai, China) by free drinking (equivalent to 6.3 times the amount of a 60 kg adult) whereas the control group mice were fed with routine water. The treatment lasted for 8 weeks. Then, the mice were put into anesthesia by intramuscular injection of Zoletil 50 (Virbac, France) and liver tissues were collected into biopsy boxes. Mice were put to death by neck broken.
2.2. Liquid chromatography-mass spectrometry-based metabolomics 25 mg of live tissue sample was weighed into a centrifuge tube and 500 µl of extract solution (acetonitrile:methanol:water = 2:2:1) was added. After 30s of vortex, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in ice-water bath. The homogenization and sonication cycle was repeated 3 times. Then, the samples were incubated at -40 °C. 400 µl of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37 °C. Next, the dried samples were reconstituted in 100 µl of 50% acetonitrile by sonication on ice for 10 min. The constitution was then centrifuged at 12000 rpm for 15 min at 4 °C and 75 µl of the supernatant was transferred to a fresh glass vial for liquid chromatography-mass spectrometry (LC/MS) analysis. The quality control (QC) sample was prepared by mixing an equal volume of aliquots of the supernatants from all of the samples.

Preprocessing of raw data
Missing data in the raw data was rst imputed using the half minimum method [15]. Next, individual peaks with > 50% null value in the peak area data and relative standard deviation (RSD) of > 20% were removed [16]. Lastly, the normalization of the data was performed with respect to the total ion current.

Statistical analysis
Statistical analysis was performed using SIMCA v15.0.2 (Sartorius Stedium Data Analytics AB, Umea, Sweden). First, multivariable analyses using principal component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) was applied to the preprocessed data.
The OPLS-DA model was veri ed using a 7-fold cross-validation technique and a permutation test (200 iterations) was performed to evaluate the tting of the model [16]. The variable importance in the projection (VIP) score was calculated for each metabolite. Next, univariable t-test was used to compare the metabolite levels across the two groups. Hierarchical clustering was performed by calculating the Euclidean distance matrix and clustered using the complete linkage method. Lastly, Pearson's correlation coe cient r was calculated for the difference of the metabolite levels between the two groups. A P value < 0.05 was considered statistically signi cant.

KEGG pathway and network analysis
First, signi cantly regulated metabolites were annotated using various databases that include the Human Metabolome Database (HMDB), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Pubchem. After annotations, all the pathways that contain the signi cantly regulated metabolites were identi ed using the Mus musculus KEGG pathway database. In order to reveal pathways that were highly correlated to GCNY treatment, enrichment analysis and topology analysis were carried out to nd the P value and impact value, respectively. A pathway's impact value was evaluated as the total importance measures of the matched metabolites divided by the total importance measures of all metabolites in each pathway [18].

Identi cation of Signi cantly Regulated Metabolites
Preprocessing of the raw data revealed a total of 126 metabolites peaks as shown in Table S1. Unsupervised PCA showed the clustering and almost total separation of the treatment and control group with no outlier present ( Figure 1A). Figure 1B shows the supervised tted model of OPLS-DA and it can be seen that the separation of the two groups was much more distinct with an R 2 X, R 2 Y and Q 2 of 0.332, 0.977 and 0.590, respectively, indicating the model has good predictability [18]. After 200 permutations, the R 2 Y and Q 2 values were generally lower than that of the original model and the Q 2 intercept value was < 0 at (0, -0.69) ( Figure 1C). These showed that there was no over tting for the OPLS-DA model [20,21]. Based on the criteria of t-test P value < 0.05 and VIP > 1 [20,21], 6 signi cantly up-regulated and 11 signi cantly down-regulated metabolites were identi ed ( Figure 1D and Table 1). Hierarchical clustering of the 17 signi cantly regulated metabolites further validated the speci c up-or downregulatory effects of GCNY in the treatment group as compared to the control group (Figure 2A). Each point represents a metabolite pro le of a biological replicate. All points fall within the Hotelling's T2 ellipse (95% con dence interval). PC [1] and PC [2]: principal component 1 and 2. (B) OPLS-DA score scatter plot of metabolite pro les in control mice and mice treated with GCNY. R 2 X, R 2 Y and Q 2 were 0.332, 0.977 and 0.590, respectively. Each point represents a metabolite pro le of a biological replicate.

Metabolic Pathway and Integrated Network Analysis
Utilizing HMDB, Pubchem and KEGG, all the pathways involving the signi cantly regulated metabolites were identi ed and are shown in Figure S1. Among these pathways, 11 metabolic pathways were associated with treatment using GCNY ( Figure 3 and Table 2) with a high correlation. A combined consideration of the P and impact value revealed that 6 pathways were signi cantly altered: 1) alanine, aspartate and glutamate metabolism, 2) pyrimidine metabolism, 3) thiamine metabolism, 4) amino sugar and nucleotide sugar metabolism, 5) pantothenate and CoA biosynthesis and 6) citrate cycle (TCA cycle). Figure 4 shows an integrated metabolic network that connects 4 of the 6 signi cantly altered pathways (pantothenate and CoA biosynthesis, pyrimidine metabolism, alanine, aspartate and glutamate metabolism and TCA cycle). Three metabolites: pantothenate, succinate and uridine were found to be down-regulated (fold change of 0.40, 0.53 and 0.64, respectively) following GCNY treatment whereas only N-carbomoyl-L-aspartate, which was involved in both the alanine, aspartate and glutamate metabolism and pyrimidine metabolism pathways, was found to be signi cantly up-regulated with a fold change of 2.72.  The x-axis represents the impact value in topology analysis whereas the y-axis represents the P value in enrichment analysis. Signi cantly altered pathways are labeled.

Discussion
The relationship between metabolism and in ammation has been profoundly investigated in recent decades. Although it is not yet known whether metabolic changes are a consequence of disease or whether primary changes to cellular metabolism might underlie or contribute to the pathogenesis of earlystage disease, changes to the metabolic pro les were observed in various diseases relating to in ammation and this includes RA as well [23]. Metabolic perturbations have been associated with RA whereby the hallmark swelling and heat observed in the joints of RA patients is considered to be a consequence of metabolic alterations. Daily whole-body resting energy expenditure is 8% higher in RA patients as compared to healthy individuals and this suggests that these metabolic changes in RA are signi cant and systemic in nature [23]. Furthermore, the catabolic condition "cachexia" occurs in RA patients with muscle atrophy and increase in body fat is associated with systemically elevated levels of proin ammatory cytokines such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), leukocyte inhibitory factor, interferon-γ (IFN-γ), and IL-6 [23]. Regulation on metabolism, for example, by inhibiting glucose metabolism exerted anti-in ammatory effects on immune cells [25] and therapeutic targets for RA from the metabolic aspect has been proposed [23].
In this study, the possible therapeutic mechanism of GCNY on RA was investigated. One of the metabolic pathways GCNY altered was the TCA cycle via the down-regulation of succinate. Succinic acid is metabolized by body cells and has a role in the tricarboxylic acid cycle as a cycle media component. It also functions as an in ammatory signaling molecule which is elevated in animals subjected to metabolic and in ammatory diseases by inducing IL-1β and HIF-1 [26]. In the context of RA, the induction of HIF-1 by succinic acid in turns lead to an induction of VEGF that will promote synovium angiogenesis [27]. It was also observed that the intra-articular microvascular blood ow has a high correlation with clinical synovitis in patients with RA [28], further suggesting that angiogenesis plays a signi cant role in the disease progression of RA. Thus, the reduction of succinic acid and angiogenesis by GNCY in RA patients might be one possible mechanism of action.
Surprisingly, uridine in the pyridine metabolism pathway was found to be downregulated in the GCNY treatment group. One of the hallmarks of RA -hyperplasia of the synovial lining layer, is caused by the excessive recruitment and accumulation of leukocytes in the synovium [29,30]. The recruited leukocytes in turn will release pro-in ammatory cytokines which activate and stimulate the proliferation of resident synoviocytes [31]. Uridine was reported to have anti-in ammatory effects in an animal model of lung in ammation [32] as well as RA by suppressing extravasation of neutrophil, macrophage and T cells into the synovium and inhibited synovial expression of intercellular adhesion molecule 1 (ICAM-1), CD-18 and cytokine production [31]. The results from this study suggest that GCNY has a component that is detrimental to RA but this negative effect is less in magnitude than the positive effect due to the reduction of angiogenesis. Further research can be done to identify the component that leads to the downregulation of uridine and the e cacy of GCNY in treating RA with and without that particular component can then be compared. Besides uridine, N-carbamoyl-L-aspartate was found to be upregulated in this pathway as well. Since there was no signi cant correlation between N-carbamoyl-Laspartate and uridine ( Figure 2B), it suggests that the downregulation of uridine was independent of the change in N-carbamoyl-L-aspartate level.
Another metabolic pathway that was found to be signi cantly altered was the pantothenate and CoA biosynthesis pathway in which pantothenate was downregulated after treating with GNCY. Early research has shown that the level of pantothenate in RA patients was lower as compared to healthy people and the decrement of pantothenate level correlated with the severity of RA [33]. There exists weak evidence for the e cacy of pantothenate in treating RA whereby a 2 g daily intake of calcium pantothenate reduced morning stiffness, pain and disability of RA patients [34]. However, the small sample size used in this study nor any further large scale, prospective study that has been performed still leave the therapeutic link between pantothenate and RA a debatable manner. The results of this study do not seem to support this claim.
Some of the other signi cant regulated metabolites are reported to be related to anti-in ammation. For example, creatinine and anserine, both upregulated in the GCNY treatment group, are attributed to antiin ammatory actions [35] with the latter being able to alleviate thioacetamide-induced brosis [36]. On the other hand, some anti-in ammatory agents were found to be reduced in the liver after GCNY treatment. One of these downregulated metabolites was thiamine. Thiamine de ciency can result in the impairment of oxidative metabolism, excitotoxicity and in ammation in which the consequences include a series of events that set the stage for cerebral vulnerability [37]. Besides that, low circulation of pyridoxal was reported to be associated with the elevation of the in ammation marker C-reactive protein [38] and being a risk factor for in ammatory-related diseases including thrombosis and in ammatory bowel disease [39]. Gamma-L-Glutamyl-L-valine exhibits antisepsis activity by reducing the expression of pro-in ammatory cytokines TNF-, IL-6, and IL-1β in the plasma and small intestine as well as inhibiting the phosphorylation of the signaling proteins c-Jun N-terminal kinases (JNK) and nuclear factor-κB inhibitory factor (IκB ) in a mouse model of LPS-induced sepsis [40]. These metabolites are not currently not associated with the pathogenesis or progression of RA but this study prompts further research to be done on investigating the relationship between these metabolites and RA to elucidate the underlying complex network mechanism.

Conclusions
In conclusion, this study validated that GCNY treatment could change the metabolic pro les in the liver of hTNF-α transgenic arthritic mouse model. Alteration to the TCA cycle and pyrimidine metabolism pathway via the regulation of succinic acid and uridine, respectively, suggested GCNY can affect the angiogenesis and leukocyte extravasation processes in RA. Further studies should be carried out to illustrate the clear action mechanisms and explore other possible metabolic targets of RA. • Consent to publish Not applicable.
• Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  The metabolome view map of signi cantly altered pathways related to treatment with GCNY. The x-axis represents the impact value in topology analysis whereas the y-axis represents the P value in enrichment analysis. Signi cantly altered pathways are labeled.