This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Exploring anti-nonalcoholic fatty liver disease mechanism of Gardeniae Fructus by combining an animal model with network pharmacology and molecular docking
Zhengxiang Xia
Tongji university
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9042-9772
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Gardeniae Fructus (GF), a traditional Chinese medicine in clinic for the treatment of nonalcoholic fatty liver disease (NAFLD). However, the mechanisms of action of GF was still margin. To explore the efficacy and mechanism of action of GF for the treatment of NAFLD, we proposed a strategy combined in vivo efficacy verification, network pharmacology analysis, molecular docking, and validity assay of target protein.
Methods
Firstly, an animal model induced by the high fat diet feed was established, then orally administrated with GF, the mRNA expression levels of lipogenesis was performed by RT-PCR, the liver tissue specimens were stained by hematoxylin and eosin (H&E), then observed by light microscopy. Secondly, network pharmacology studies clarified the relationship among the active constituents, target protein, and pathways, and then explored by the molecular docking. Finally, validity assay of target protein was performed in surface plasmon resonance (SPR) test.
Results
GF protected against NAFLD in rats. Network pharmacology showed that quercetin, oleanolic acid, and geniposide, targeted on PPARα, PPARγ, and CA2 genes, through regulating PPAR, AMPK, and cGMP-PKG signal pathways, to protect against NAFLD. And the
Conclusion
GF could alleviate NAFLD through the molecular mechanisms explored by network pharmacology, molecular docking, and surface plasmon resonance, those method can be effective tools to clarify the mechanisms of actions of traditional Chinese medicine from a holistic perspective.
Keywords
Gardeniae Fructus, nonalcoholic fatty liver disease, network pharmacology, molecular docking, surface plasmon resonance
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Quility control of GF
The GF was purchased from Shanghai Kang Qiao Herbal Pieces Co. Ltd. (Shanghai, China). HPLC was used to identify the active components of GF. And the compound was finally confirmed by the comparison with the authentic compound. The chromatographic separation was carried out on a Diamonsil C18 column (150 × 4.6 mm I.D., 5 mm) at 25 ℃. The mobile phase consisted of acetonitrile (solvent A) and water (0.1% formic acid) (solvent B). The optimized elution condition was applied as follows: 0–60 min, 5–95% A. The solvent flow rate and injection volume was kept as 0.5 mL/min and 5 µL, respectively. Then the result was exhibited (Fig. 2), compounds 1–4 were identified as geniposide, genipin 1-gentiobioside, 6α-hydroxygeniposide, and gardenoside, respectively.
Regulation of the genes involved in lipogenesis and fat oxidation by GF
NAFLD is a chronic disease affecting liver tissues that characterized by an increasing the mRNA expression of lipogenesis (SREBP-1c, FAS, SCD-1, CD36, PPAR-α, and CPT-1). As shown (Fig. 3), they were significantly reduced following the supplement of GF on HFD-fed rats compared to the non-treatment HFD-fed rats, and also in a dose-dependent manner. Furthermore, the high dosage (100 mg/kg) of GF administrated on the HFD-fed rats showed comparative effects to the positive control drug metformin owing to they had similar values of mRNA expression of lipogenesis. These data suggested that GF protected against NAFLD through regulating the mRNA expression of lipogenesis in liver tissue.
GF reduced lipids, liver Enzymes, and MDA Levels in serum
To further explore the preventive effects of GF on HFD-induced liver steatosis, we measured the serum biochemical indices in different groups of rats. As exhibited (Fig. S1), the serum TC, LDL-C, and TG levels significantly increased in the model rats fed a HFD diet compared with the rats fed a normal diet. However, while the HFD-fed rats orally administrated with GF, serum TC, LDL-C, and TG levels in those rats decreased significantly, and also in a dose-dependent manner. The serum ALT, AST, and LDH levels (hepatic damage markers) exhibited the similar trends. Besides, serum MDA levels, the products of lipid peroxidation, an oxidative marker used as an indicator of oxidative damage, also showed the similar trends. Therefore, the results showed that GF had comparative effects to metformin through resisting oxidative stress and reducing serum lipids.
GF decreased serum glucose and insulin in HFD-fed rat
Serum glucose and insulin levels are important biomarkers in metabolic diseases especially in NAFLD. They changed in different groups of rats as displayed (Fig. S2), the serum glucose and insulin levels were increased in HFD-induced rats compared to the control rats, whereas they reduced after intragastric administration on the HFD-fed rats with GF obviously compared with the HFD-fed rats without any treatments. Moreover, the high dosage (100 mg/kg) of GF treatment showed the comparative effects to the positive control metformin against the HFD-induced rats with NAFLD. Collectively, GF may protect against NAFLD by reducing serum glucose and insulin levels.
GF improved the liver steatosis in HFD-fed model rats
Liver fat accumulation during the ingestion of HFD can lead to NAFLD. As shown (Fig. S3), the histological evaluation of liver samples exhibited obviously increases in lipid droplets in hepatocytes in the HFD-fed rats compared those without any treatment, while these lipid droplets were reduced significantly following the GF and positive control metformin supplement on HFD-fed groups. In the other side, according to the above biochemical data analysis, liver lipid contents, liver TC, and TG levels in the HFD group were significantly increased compared with those in the control group, which was consistent with the histological data. Thus, GF showed the similar effects to the positive control metformin for the treatment of NAFLD.
In silico network analysis and prediction of target genes and pathways related to NAFLD
Several compounds in GF may play a synergistic manner to protect against NAFLD. To further illustrate which ingredients from GF may attribute to the hepatoprotective effects, a network pharmacology experiment was carried out. According to the analysis of this network, seven components in Table 2 were selected and linked to 15 potential target genes were present (Fig. 4). Among these compounds, quercetin (7), oleanolic acid (3), kaempferol (3), and geniposide (3) were linked to three or more genes with higher degrees. In addition, the core genes including peroxisome proliferator-activated receptor alpha (PPARα) regulated by oleanolic acid and quercetin, Peroxisome proliferator-activated receptor gamma (PPARγ) targeted by quercetin and kaempferol, and carbonic anhydrase II (CA2) linked by genipin and geniposide. In all, the above compounds and genes might play myriad roles in the NAFLD development and progression.
Due to technical limitations, Table 2 is provided in the Supplementary Files section.
PPI Network analysis
Through the analysis of the interaction between target genes, the result was shown (Fig. S4), proliferator-activated receptor alpha (PPARα), Peroxisome proliferator-activated receptor gamma (PPARγ) and carbonic anhydrase II (CA2) have greater degrees than others, which indicated that these proteins might play important roles in NAFLD. Interestingly, it was consisted with the conclusion from the above network analysis.
To understand the hepatoprotective mechanism of GF against NFALD, we performed functional enrichment analysis of target genes (Fig. 5) of bio-active compounds from GF using DAVID software and the KEGG database, potential target genes were functionally associated with various signal transduction pathways such as peroxisome proliferator-activated receptor (PPAR), glucagon signaling pathway, AMPK signaling pathway (AMPK) and cGMP-PKG signaling pathway (cGMP-PKG) shown in Table 3. Interestingly, many of the potential target genes appeared to be connected to the PPAR signaling pathway. Subsequently, GF protected against NAFLD through multiple pathways especially the PPAR signaling pathway.
| Pathway Classification | Pathway ID | Term | Target Gene |
|---|---|---|---|
| Signal transduction | hsa03320 | PPAR signaling pathway | PPARA, PPARD, PPARG |
| Signal transduction | hsa04970 | Salivary secretion | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04080 | Neuroactive ligand-receptor interaction | ADRB2, ADRA1B, ADRA1A, GLP1R |
| Signal transduction | hsa04922 | Glucagon signaling pathway | GCG, PPARA, ACACA |
| Signal transduction | hsa04152 | AMPK signaling pathway | PPARG, ACACA, ADRA1A |
| Signal transduction | hsa04261 | Adrenergic signaling in cardiomyocytes | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04022 | cGMP-PKG signaling pathway | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04020 | Calcium signaling pathway | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04024 | cAMP signaling pathway | PPARA, ADRB2, GLP1R |
Docking exercises of binding the main ingredients and protein
Computational docking exercises were conducted to mimic the characteristic of ingredient-target binding mode. The results showed that oleanolic acid and quercetin formed stronger or comparative interactions with PPARα to the native ligand (Fig. 6) featuring higher or similar docking score. The results were similar to genipin and geniposide formed interactions with CA2, and quercetin and kaempferol formed interactions with PPARγ. Taken together, GF protected against NAFLD via multi-compounds regulating multi-targets, for example, oleanolic acid and quercetin targeted on PPARα, genipin and geniposide targeted on CA2, quercetin and kaempferol targeted on PPARγ.
Validity Assay of Target Protein
Quercetin and PPARγ were selected to take part in the SPR test, The results showed that they had a high response and affinity with the Kd value of 71.5 µM (Fig. S5).
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 13 May, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Learn more about our company.
This is a preprint. It has not completed peer review.
Research
Exploring anti-nonalcoholic fatty liver disease mechanism of Gardeniae Fructus by combining an animal model with network pharmacology and molecular docking
Zhengxiang Xia
Tongji university
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9042-9772
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 13 May, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Gardeniae Fructus (GF), a traditional Chinese medicine in clinic for the treatment of nonalcoholic fatty liver disease (NAFLD). However, the mechanisms of action of GF was still margin. To explore the efficacy and mechanism of action of GF for the treatment of NAFLD, we proposed a strategy combined in vivo efficacy verification, network pharmacology analysis, molecular docking, and validity assay of target protein.
Methods
Firstly, an animal model induced by the high fat diet feed was established, then orally administrated with GF, the mRNA expression levels of lipogenesis was performed by RT-PCR, the liver tissue specimens were stained by hematoxylin and eosin (H&E), then observed by light microscopy. Secondly, network pharmacology studies clarified the relationship among the active constituents, target protein, and pathways, and then explored by the molecular docking. Finally, validity assay of target protein was performed in surface plasmon resonance (SPR) test.
Results
GF protected against NAFLD in rats. Network pharmacology showed that quercetin, oleanolic acid, and geniposide, targeted on PPARα, PPARγ, and CA2 genes, through regulating PPAR, AMPK, and cGMP-PKG signal pathways, to protect against NAFLD. And the
Conclusion
GF could alleviate NAFLD through the molecular mechanisms explored by network pharmacology, molecular docking, and surface plasmon resonance, those method can be effective tools to clarify the mechanisms of actions of traditional Chinese medicine from a holistic perspective.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Quility control of GF
The GF was purchased from Shanghai Kang Qiao Herbal Pieces Co. Ltd. (Shanghai, China). HPLC was used to identify the active components of GF. And the compound was finally confirmed by the comparison with the authentic compound. The chromatographic separation was carried out on a Diamonsil C18 column (150 × 4.6 mm I.D., 5 mm) at 25 ℃. The mobile phase consisted of acetonitrile (solvent A) and water (0.1% formic acid) (solvent B). The optimized elution condition was applied as follows: 0–60 min, 5–95% A. The solvent flow rate and injection volume was kept as 0.5 mL/min and 5 µL, respectively. Then the result was exhibited (Fig. 2), compounds 1–4 were identified as geniposide, genipin 1-gentiobioside, 6α-hydroxygeniposide, and gardenoside, respectively.
Regulation of the genes involved in lipogenesis and fat oxidation by GF
NAFLD is a chronic disease affecting liver tissues that characterized by an increasing the mRNA expression of lipogenesis (SREBP-1c, FAS, SCD-1, CD36, PPAR-α, and CPT-1). As shown (Fig. 3), they were significantly reduced following the supplement of GF on HFD-fed rats compared to the non-treatment HFD-fed rats, and also in a dose-dependent manner. Furthermore, the high dosage (100 mg/kg) of GF administrated on the HFD-fed rats showed comparative effects to the positive control drug metformin owing to they had similar values of mRNA expression of lipogenesis. These data suggested that GF protected against NAFLD through regulating the mRNA expression of lipogenesis in liver tissue.
GF reduced lipids, liver Enzymes, and MDA Levels in serum
To further explore the preventive effects of GF on HFD-induced liver steatosis, we measured the serum biochemical indices in different groups of rats. As exhibited (Fig. S1), the serum TC, LDL-C, and TG levels significantly increased in the model rats fed a HFD diet compared with the rats fed a normal diet. However, while the HFD-fed rats orally administrated with GF, serum TC, LDL-C, and TG levels in those rats decreased significantly, and also in a dose-dependent manner. The serum ALT, AST, and LDH levels (hepatic damage markers) exhibited the similar trends. Besides, serum MDA levels, the products of lipid peroxidation, an oxidative marker used as an indicator of oxidative damage, also showed the similar trends. Therefore, the results showed that GF had comparative effects to metformin through resisting oxidative stress and reducing serum lipids.
GF decreased serum glucose and insulin in HFD-fed rat
Serum glucose and insulin levels are important biomarkers in metabolic diseases especially in NAFLD. They changed in different groups of rats as displayed (Fig. S2), the serum glucose and insulin levels were increased in HFD-induced rats compared to the control rats, whereas they reduced after intragastric administration on the HFD-fed rats with GF obviously compared with the HFD-fed rats without any treatments. Moreover, the high dosage (100 mg/kg) of GF treatment showed the comparative effects to the positive control metformin against the HFD-induced rats with NAFLD. Collectively, GF may protect against NAFLD by reducing serum glucose and insulin levels.
GF improved the liver steatosis in HFD-fed model rats
Liver fat accumulation during the ingestion of HFD can lead to NAFLD. As shown (Fig. S3), the histological evaluation of liver samples exhibited obviously increases in lipid droplets in hepatocytes in the HFD-fed rats compared those without any treatment, while these lipid droplets were reduced significantly following the GF and positive control metformin supplement on HFD-fed groups. In the other side, according to the above biochemical data analysis, liver lipid contents, liver TC, and TG levels in the HFD group were significantly increased compared with those in the control group, which was consistent with the histological data. Thus, GF showed the similar effects to the positive control metformin for the treatment of NAFLD.
In silico network analysis and prediction of target genes and pathways related to NAFLD
Several compounds in GF may play a synergistic manner to protect against NAFLD. To further illustrate which ingredients from GF may attribute to the hepatoprotective effects, a network pharmacology experiment was carried out. According to the analysis of this network, seven components in Table 2 were selected and linked to 15 potential target genes were present (Fig. 4). Among these compounds, quercetin (7), oleanolic acid (3), kaempferol (3), and geniposide (3) were linked to three or more genes with higher degrees. In addition, the core genes including peroxisome proliferator-activated receptor alpha (PPARα) regulated by oleanolic acid and quercetin, Peroxisome proliferator-activated receptor gamma (PPARγ) targeted by quercetin and kaempferol, and carbonic anhydrase II (CA2) linked by genipin and geniposide. In all, the above compounds and genes might play myriad roles in the NAFLD development and progression.
Due to technical limitations, Table 2 is provided in the Supplementary Files section.
PPI Network analysis
Through the analysis of the interaction between target genes, the result was shown (Fig. S4), proliferator-activated receptor alpha (PPARα), Peroxisome proliferator-activated receptor gamma (PPARγ) and carbonic anhydrase II (CA2) have greater degrees than others, which indicated that these proteins might play important roles in NAFLD. Interestingly, it was consisted with the conclusion from the above network analysis.
To understand the hepatoprotective mechanism of GF against NFALD, we performed functional enrichment analysis of target genes (Fig. 5) of bio-active compounds from GF using DAVID software and the KEGG database, potential target genes were functionally associated with various signal transduction pathways such as peroxisome proliferator-activated receptor (PPAR), glucagon signaling pathway, AMPK signaling pathway (AMPK) and cGMP-PKG signaling pathway (cGMP-PKG) shown in Table 3. Interestingly, many of the potential target genes appeared to be connected to the PPAR signaling pathway. Subsequently, GF protected against NAFLD through multiple pathways especially the PPAR signaling pathway.
| Pathway Classification | Pathway ID | Term | Target Gene |
|---|---|---|---|
| Signal transduction | hsa03320 | PPAR signaling pathway | PPARA, PPARD, PPARG |
| Signal transduction | hsa04970 | Salivary secretion | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04080 | Neuroactive ligand-receptor interaction | ADRB2, ADRA1B, ADRA1A, GLP1R |
| Signal transduction | hsa04922 | Glucagon signaling pathway | GCG, PPARA, ACACA |
| Signal transduction | hsa04152 | AMPK signaling pathway | PPARG, ACACA, ADRA1A |
| Signal transduction | hsa04261 | Adrenergic signaling in cardiomyocytes | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04022 | cGMP-PKG signaling pathway | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04020 | Calcium signaling pathway | ADRB2, ADRA1B, ADRA1A |
| Signal transduction | hsa04024 | cAMP signaling pathway | PPARA, ADRB2, GLP1R |
Docking exercises of binding the main ingredients and protein
Computational docking exercises were conducted to mimic the characteristic of ingredient-target binding mode. The results showed that oleanolic acid and quercetin formed stronger or comparative interactions with PPARα to the native ligand (Fig. 6) featuring higher or similar docking score. The results were similar to genipin and geniposide formed interactions with CA2, and quercetin and kaempferol formed interactions with PPARγ. Taken together, GF protected against NAFLD via multi-compounds regulating multi-targets, for example, oleanolic acid and quercetin targeted on PPARα, genipin and geniposide targeted on CA2, quercetin and kaempferol targeted on PPARγ.
Validity Assay of Target Protein
Quercetin and PPARγ were selected to take part in the SPR test, The results showed that they had a high response and affinity with the Kd value of 71.5 µM (Fig. S5).