Effects of active, inactive, and derivatives of Akkermansia muciniphila on the expression of the endocannabinoid system and PPARs genes

This study aimed to investigate the effects of active and heat-inactivated forms of Akkermansia muciniphila, bacterium-derived outer membrane vesicles (OMVs), and cell-free supernatant on the transcription of endocannabinoid system (ECS) members, including cannabinoid receptors 1 and 2 (CB1 and CB2), fatty acid amide hydrolase (FAAH), and peroxisome proliferator-activated receptors (PPARs) genes (i.e., α, β/δ, and δ) in Caco-2 and HepG-2 cell lines. After the inoculation of A. muciniphila in brain heart infusion enriched medium, OMVs and cell-free supernatant were extracted. For the investigation of the effects of bacteria and its derivatives on the expression of ECS and PPARs genes, the aforementioned cells were treated by active and heat-inactivated bacteria, OMVs, and cell-free supernatant. Quantitative real-time polymerase chain reaction analysis revealed that both forms of the bacterium, bacterial-derived OMVs, and cell-free supernatant could affect the expression of CB1, CB2, FAAH, and PPARs genes (i.e., α, β/δ, and δ) significantly (P < 0.05). Considering the engagement of the aforementioned genes in metabolic pathways, it might be suggested that both forms of the bacterium, OMVs, and cell-free supernatant might have the potential to serve as a probiotic, paraprobiotic, and postbiotic candidate to prevent obesity, metabolic disorders, and liver diseases.

www.nature.com/scientificreports/ as a confirmation for the presence and sizes of OMVs. The sample was prepared by negative staining and then observed by PHILIPS (Netherlands) EM 208.
RNA extraction and cDNA synthesis. The cells were collected from six wells (5 × 10 5 cells per well), and their total RNA was extracted using RNX-Plus Solution (2000 ng/μl; Sinacolon, Iran). In order to purify the extracted RNAs, they were treated by DNAse for 1 h at 37 °C. The presence and quality of the extracted RNAs were checked out by agarose gel electrophoresis and spectrophotometry using NanoDrop 2000 (Thermo Fisher Scientific, USA). After balancing the concentrations of RNAs, complementary deoxyribonucleic acid (cDNA) was synthesized using a cDNA synthesis kit (Parstous, Iran) according to manufacturers' instructions. A no reverse transcriptase control cDNA was included in qRT-PCR analysis. The concentrations were checked out by spectrophotometry using a Nanodrop device 47  Statistical analysis. The data extracted from qRT-PCR were analyzed by GraphPad Prism software (version 8.4.3; GraphPad Software Inc., San Diego, CA, USA) using an independent sample t-test (between two groups) and one-way analysis of variance (with Tukey's multiple comparison test to estimate differences between means which was used to compare means among more than two groups for each parameter). All results were considered statistically significant with a p-value less than 0.05 12,47 . qRT-PCR analysis of the associated genes expression in Caco-2 cell line. Effects of active and inactivated A. muciniphila, derived OMVs, and cell-free supernatant on transcription level of the studied genes Involved in endocannabinoid system. Data analysis showed a significant decrease in the messenger ribonucleic acid (mRNA) level of the CB1 receptor by MOI 10 of active A. muciniphila (P = 0.04) and both 50 and 100 μg/ mL concentrations of OMVs (P = 0.01) (Fig. 2). All three MOIs of 10, 50, and 100 of the inactivated form of the bacterium could remarkably (P = 0.0008, P = 0.0001, and P = 0.0006, respectively) increase the mRNA level of the CB1 receptor; nevertheless, the cell-free supernatant did not have a significant effect on the expression of the CB1 gene (P = 0.053). A noticeable decrease occurred in the mRNA level of the CB2 receptor by MOI 100 of active A. muciniphila, 100 μg/ml concentrations of OMV, and concentration of 7% (v/v [medium/cell-free supernatant]) cell-free supernatant (P = 0.01, P = 0.03, and P = 0.04, respectively); however, the MOIs of 10 and 50 of the inactivated form of the bacterium increased the mRNA level of the CB2 receptor significantly (P = 0.0007 and P = 0.01, respectively) (Fig. 2).
Effects of the above-mentioned treatments on transcription level of PPARs (i.e., α, β/δ, and ϒ) genes. In reference to The MOIs 50 and 100 of active A. muciniphila (P = 0.004 and P < 0.0001, respectively), MOI 10 of the inactivated form of the bacterium (P = 0.03), and both 50 and 100 μg/mL concentrations of OMVs (P = 0.012 and P = 0.007, respectively) significantly increased the mRNA level of PPARϒ gene; nevertheless, the cell-free supernatant could not affect its expression significantly (P = 0.79; Fig. 3).
qRT-PCR analysis of the associated genes expression in HepG-2 cell line. Effects of active and inactivated A. muciniphila, derived OMVs, and cell-free supernatant on transcription level of the studied genes involved in endocannabinoid system. Data analysis showed that only MOI 50 of the active form of the bacterium significantly decreased the CB1 receptor transcription level (P = 0.033), and none of the inactive bacteria, OMVs, and cell-free supernatant could affect the transcription level of the CB1 receptor (P > 0.05; Fig. 4).
According to the results shown in Fig. 4, 100 μg/mL concentrations of OMVs and 7% (v/v [medium/cell-free supernatant]) concentration of cell-free supernatant remarkably increased the mRNA level of the CB2 receptor (P = 0.0002 and P = 0.0034, respectively); nonetheless, neither active nor inactive forms of the bacterium affected its transcription level (P > 0.05). Only 100 μg/mL concentrations of OMVs increased FAAH mRNA level significantly (P = 0.013), and none of the concentrations of the active and inactive forms and cell-free supernatant affected it (P > 0.05; Fig. 4).
Effects of the above-mentioned treatments on transcription level of PPARs (i.e., α, β/δ, and ϒ) genes. The MOI 10 of the active form of the bacterium, 100 μg/mL concentrations of OMVs, and 7% (v/v [medium/cell-free supernatant]) concentration of cell-free supernatant noticeably increased the transcriptome level of the PPARα gene (P = 0.01, P = 0.009, and P = 0.029, respectively); however, the inactive form of the bacterium did not affect it (P > 0.05; Fig. 5).
As shown in Fig. 5, MOI 10 of the active form and 100 μg/mL concentrations of OMVs could remarkably increase the mRNA level of the PPARβ/δ gene (P = 0.032 and P = 0.0005, respectively); nevertheless, neither inactive form nor the cell-free supernatant affected its transcription level (P > 0.05).
Finally, there was a significant increase in the mRNA level of the PPARϒ gene only at MOI 10 and 50 treatments of the active form of the bacterium (P = 0.034 and P = 0.047, respectively), and none of the inactive form, OMVs, and cell-free supernatant affected it (P > 0.05; Fig. 5).

Discussion
A. muciniphila is an anaerobic Gram-negative bacterium that appropriates about 3-5% of the gut bacteria in healthy humans 12,14 . The evidence has confirmed its role in gut barrier regulation and its involvement in metabolic and homeostatic procedures 4,17 . Recently, a great interest has been attracted on this subject due to its potential of introducing as the next generation probiotics 12 . A. muciniphila mainly colonizes in the gut, and due to some leaky gut conditions, the bacterium and related derivatives 52 might pass through the gut barrier and enter some other organs, such as the liver through the gut-liver axis 4,10,11 . This might happen based on the interaction between the ECS, gut microbiota, and the liver 25 , and the involvement of PPARs genes in the regulation of gut barrier  (2) [42][43][44] . Therefore, this study investigated the effects of A. muciniphila and its derivatives on ECS-related and PPARs genes in Caco-2 and Hep-G2 cell lines in parallel. The ECS consists of three main compartments, including cannabinoids, cannabinoid receptors, and metabolic enzymes, concentrated mainly in the brain and some peripheral tissues. They play various roles, such as involvement in the regulation of hunger and satiety, relaxation, protection, immunity, metabolism, decreasing inflammation, and increasing permeability in the GIT. It also influences some diseases, such as obesity, type 2 diabetes, steatosis, fibrogenesis, and alcoholic and nonalcoholic liver diseases, mainly through CB1 function [25][26][27][28]53 .
Regarding the important role of A. muciniphila in gut barrier integrity and decreasing permeability which prevents metabolic disorders associated with obesity 54,55 , the present study showed that MOI 10 of A. muciniphila and both concentrations of 50 and 100 μg/mL of A. muciniphila-derived OMVs could decrease the level of CB1 mRNA in Caco-2 cells which promotes the idea of using the active A. muciniphila as a probiotic candidate and bacterial-derived OMVs for CB1 expression regulation to prevent metabolic disorders associated with obesity.
This study demonstrated that all MOIs (i.e., 10, 50, and 100) of the inactivated form of the bacterium increased the CB1 mRNA level in Caco-2 cells. Consistent with the results of the present study, Everard et al. (2013) observed that in spite of the active bacterium, heat-killed A. muciniphila could not improve the thickness of    61 . Therefore, FAAH function has a reverse relationship with cannabinoid receptors. The current study data showed that MOI 50 and 100 of active A. muciniphila, both concentrations of OMV 50 and 100 μg/ml, and cell-free supernatant could significantly increase the level of FAAH mRNA in Caco-2 cells.
The MOI 10, 50, and 100 of inactive bacteria also increased FAAH mRNA significantly. Probably, there is a modulatory function of FAAH, since Murakami et el. (2007) discussed that bacterial lipopolysaccharide (LPS) as one of the magic components of MAMPs 19 in A. muciniphila 62 induces the production of anandamide which is mentioned as a ligand for the CB1 receptor. Meanwhile, the LPS stimulation could not affect FAAH; therefore, it seems natural that the rate of FAAH mRNA increased by more anandamide production to control the overproduction of these ligands 63 . Considerably, there is a reverse relationship in the treatment results of MOI 50 of inactive A. muciniphila between CB1 and FAAH in Caco-2 cells. www.nature.com/scientificreports/ The PPARs belong to the nuclear receptor superfamily serving as transcription factors that regulate numerous transcriptional activities, such as metabolic, inflammatory, and developmental processes. The PPARs are composed of three isotypes, including PPARα, PPARβ/δ, and PPARϒ, which have different distributions in the human body; however, all three are highly expressed in the colon. The evidence has shown that there is a direct interaction between gut microbiota and PPARs in a way that gut microbiota might induce PPARs expression and activation 40,64 . In 2011, Goto et al. reported that PPARα stimulates fatty acid oxidation in adipocytes and ameliorates metabolic disorders 65   www.nature.com/scientificreports/ tissue 54,67,68 . Consistent with these findings, we observed that MOIs 50 and 100 of the active form, MOIs 10 and 50 of the inactivated form, both 50 and 100 μg/mL concentrations of OMVs, and cell-free supernatant could significantly increase the rate of PPARα transcriptome. The PPARβ mRNA level was almost increased by the same treatments significantly by the effects of MOI 50 of the active and MOI 10 of inactivated forms and OMV 100 μg/mL. The result received for PPARϒ was in the same direction as previous isotypes in such a way that mRNA level was increased significantly by MOIs 50 and 100 of the active and MOI 10 of the inactivated forms and both 50 and 100 μg/mL concentrations of OMVs. All these results confirmed the positive effects of A. muciniphila and related derivatives on the transcription of PPARs genes, probably through affecting fatty acids oxidation and energy metabolism.
In gut microbiota overgrowth conditions or tight junctions' impairments, the integrity of the gut decreases, which leads to increasing the gut permeability and translocation of bacteria from the gut lumen to the portal and/ or systemic circulation. In such conditions, the bacteria might enter the liver through the gut-liver axis 11 . In this study, MOI 50 of active A. muciniphila decreased the mRNA level of the CB1 receptor significantly in Hep-G2 cell lines; nevertheless, inactivated A. muciniphila, OMVs, and cell-free supernatant could not affect CB1 receptor transcription. Active A. muciniphila induces beneficial effects on hepatocytes through the downregulation of the CB1 receptor since Mallat et al. in 2013 explained that the CB1 receptor is expressed in hepatocytes and hepatic myofibroblasts and involved in numerous liver diseases, such as alcohol-induced liver disease, nonalcoholic fatty liver disease, fibrogenesis, and cardiovascular alterations associated with cirrhosis 43 .
Studies have shown that the expression of CB2 receptors in hepatocytes is modest, mainly contributes to hepatoprotective, anti-inflammatory, antioxidant, and immunomodulatory effects 69,70 , and plays a prohibiting role in liver fibrosis and alcohol-induced liver damage, compared to CB1 receptor 28,43,71 . In this study, neither active nor inactivated A. muciniphila could affect CB2 receptor expression; however, 100 μg/mL concentration of OMVs and cell-free supernatant noticeably increased the CB2 expression in mRNA level representing the more influence of the bacterium derivatives and metabolites rather than the bacterium itself. These results might have the root in A. muciniphila-derived OMVs and related cell-free supernatant that might preserve as therapeutic agents to improve liver health conditions. Moreover, it was observed that none of the treatments had an influence on FAAH expression except 100 μg/mL concentration of OMVs. This finding might be considered satisfactory since FAAH is a hydrolyzing enzyme for the ligands of both receptors and naturally balances the ECS-dependent health of the liver.
A. muciniphila produces SCFAs, such as propionate, butyrate, and acetate 54 which can activate PPAR α expression through which prevents lipid accumulation in the liver 72 . The PPARα is mostly found in hepatocytes, plays a critical role in fatty acid uptake and fatty acid oxidation, decreases the production of very-low-density lipoprotein, and increases high-density lipoprotein. It also downregulates the hepatic inflammatory processes 32 . In the current study, MOI 10 of the active A. muciniphila, 100 μg/mL concentration of OMVs, and the-cell free supernatant considerably increased the PPARα expression in mRNA level. This result suggests that less amount of the active bacterium might promote PPARα expression better than higher amounts which might cause no inflammation in liver tissues. Similar results have been obtained for PPARβ/δ as it was observed that MOI 10 of the active A. muciniphila and 100 μg/mL concentration of OMVs significantly increased the mRNA level of PPARβ/δ.
Previous studies revealed that the potential role of PPARβ/δ in hepatocytes is apparent; they assumed that this gene is highly expressed in these cells and regulates glucose utilization and fatty acid metabolism. It also participates in the alleviation of inflammation and fibrosis 32,72,73 . Transcription analysis showed that A. muciniphila's MOIs of 10 and 50 could enhance the PPARϒ transcriptome level, which is according to the results of studies of several researchers who concluded that gut microbiota is associated with metabolism, including PPARs expression related pathways 72,74 . Wagnerberger et al. in 2013 reported that intake of Lactobacillus casei upregulated hepatic PPARϒ leading to inhibition of Toll-like receptor 4 and suppression of steatosis 75 . Additionally, in previous studies in 2011, Nan et al. declared that the overexpression of PPARϒ can influence some liver diseases, such as reducing effect on steatosis, inflammation, and fibrosis in steatohepatitis murine model 76 . In a recent study, Keshavarz et al. reported that MOI 10 of heat-killed bacteria increased the expression of PPARϒ remarkably 18 . The effect of PPARϒ on fatty acid oxidation and glucose homeostasis as an insulin sensitizer was also lately reviewed by Wu et al. 72 .
In normal conditions, the bacterial derivatives might pass through the gut barrier either directly or via dynamin-dependent endocytosis and then translocate toward the liver 52 . In some leaky gut conditions, the bacteria might translocate from the gut to the liver through the gut-liver axis 10,11 . Some associations might be observed in the gene transcription of the cells in both organs. For example, as shown in Figs. 2 and 4, active A. muciniphila could decrease the level of CB1 transcription in both cell lines. Furthermore, OMVs treatments in both cell lines increased the FAAH mRNA level. The transcription of PPARα, β/δ, and ϒ was increased by active A. muciniphila in both cell lines (Figs. 3 and 5). The increased level of PPARα by the treatment of inactive bacteria and derived OMVs in Caco-2 cells was in line with HepG-2 cells (Figs. 3 and 5). A similar result was observed by the effect of OMVs treatments on PPARβ/δ transcription in two cell lines (Figs. 3 and 5). One contradiction was the effects of OMVs and cell-free supernatant treatments on the CB2 transcription level, which were contradictory in two cell lines. They might affect CB2 gene transcription with different mechanisms in the aforementioned cell lines, which requires more investigation.
The limitations of the current study were accomplishing these experiments in expression level and performing the same procedure under in vivo conditions, especially to investigate the correlation of gut and liver gene expressions affected by the aforementioned treatments.
In conclusion, we considered the positive effects of A. muciniphila and its derivatives, such as OMVs and bacterial metabolites, on controlling the activity of ECS compartments which might influence obesity, metabolic disorders, and liver diseases depending on their type. According to the present study results, A. muciniphila and www.nature.com/scientificreports/ its derivatives might be considered probiotic, paraprobiotic, and postbiotic candidates to protect organs against metabolic syndromes and liver diseases. It is worth mentioning that there is an interaction between eCBs and PPARs genes which introduces some possible pathways of PPARs activation by eCBs either directly or indirectly 41 . Considering the essential roles of PPARs as nuclear receptors in the regulation of energy homeostasis, metabolism, cell differentiation, and inflammation [77][78][79][80] , the evidence suggests that numerous ECB functions, such as analgesic, neuroprotective, neuronal function modulation, anti-inflammatory, metabolic, anti-tumor, gastrointestinal, and cardiovascular effects of some cannabinoids are mediated by PPARs 41 . This association attracts the attention to scrutinize all possible pathways that might influence this study's findings.

Data availability
All data that support all the experimental findings in this article is available in the Supplementary Data File provided.