Gut Microbiota-Mediated Personalized Treatment of Hyperlipidemia Using Berberine.

Nitroreductases (NRs) are bacterial enzymes that reduce nitro-containing compounds. We have previously reported that NR of intestinal bacteria is a key factor promoting berberine (BBR) intestinal absorption. We show here that feeding hamsters with high fat diet (HFD) caused an increase in blood lipids and NR activity in the intestine. The elevation of fecal NR by HFD was due to the increase in either the fraction of NR-producing bacteria or their activity in the intestine. When given orally, BBR bioavailability in the HFD-fed hamsters was higher than that in those fed with normal chow (by +72%, *P<0.05). BBR (100 mg/kg/day, orally) decreased blood lipids in the HFD-fed hamsters (**P<0.01) but not in those fed with normal diet. Clinical studies indicated that patients with hyperlipidemia had higher fecal NR activity than that in the healthy individuals (**P<0.01). Similarly, after oral administration, the blood level of BBR in hyperlipidemic patients was higher than that in healthy individuals (*P<0.05). Correlation analysis revealed a positive relationship between blood BBR and fecal NR activity (r=0.703). Thus, the fecal NR activity might serve as a biomarker in the personalized treatment of hyperlipidemia using BBR.

Introduction

Berberine (BBR) is an alkaloid (Figure , mw 371.8) isolated from plants like Coptis chinensis, and Berberis vulgaris et al. For decades, BBR has been used as an over-the-counter (OTC) drug in China to treat bacteria-caused diarrhea and its safety in humans has been well documented 1-3. We have previously reported that BBR was effective as a drug for patients with hyperlipidemia and type 2 diabetes 2-3. The effect of BBR on blood lipids and glucose was associated with several key molecules including low-density lipoprotein receptor (LDLR), adenosine monophosphate-activated protein kinase (AMPK), insulin receptor (InsR), and proprotein convertase subtilisin/kexin type 9 (PCSK9) et al. 2-9. In the past 10 years, these results have been verified by independent researchers and clinical groups in and outside China and BBR has been approved by the Chinese FDA for clinical studies. However, the unresolved issues for BBR include, for instance, identifying biomarkers that would help in the selection of patients sensitive to BBR treatment. A previous study focusing on genetic variations in the LDLR mRNA 3'UTR region in humans failed to identify signature mutations associated with the treatment outcome of BBR 10, probably because BBR is a multi-target drug 11-12. Our previous results showed that BBR is mainly metabolized by CYP2D6, CYP1A2, and CYP3A4 in vivo 13. The low blood concentration of BBR and its pharmacokinetics have been the subject of much research 14, 15. Also, as described by Deng et al., entero-hepatic circulation might play a role in BBR pharmacokinetics 16. We have recently demonstrated that an important factor in BBR absorption in the intestine is gut microbiota, which convert BBR into its absorbable form, dihydroberberine (dhBBR) which showed a 5-10 fold higher absorption rate than BBR 17. The reduction from BBR to dhBBR is mediated through nitroreductase (NR) of gut microbiota 17. Following absorption in the intestine, dhBBR is quickly oxidized back to BBR and enter blood 17. Thus, NR in gut microbiota is critical for regulating blood levels of BBR.

NR is a group of enzymes primarily found in bacterial lineages. Members of this family utilize flavin mononucleotide (FMN) as a co-factor to catalyze the reductive reaction of the nitro-group on nitroaromatic and nitroheterocyclic compounds including chemicals with nitro functional groups, for instance the 7-position nitrogen in the BBR's structure 18. In the present study, we show that high level of NR activity in gut microbiota increased BBR oral bioavailability and might be a potential biomarker to predict the therapeutic efficacy of BBR.

Materials and Methods

Chemicals and reagents

BBR was obtained from the J&K Scientific Ltd (Beijing, China). Tetrahydropalmatine, as the internal standard, was from the National Institute for Food and Drug Control (Beijing, China). Thalifendine (M1), berberrubine (M2), demethyleneberberine (M3), jatrorrhizine (M4) and palmatine (M5), and dihydroberberine (dhBBR) were acquired from the Chengdu Herbpurity Co., Ltd (Chengdu, China). The purity of the standards was above 98% according to the high-performance liquid chromatography (HPLC) assay. Other chemical reagents from the Sino-pharm Chemical Reagent Co, Ltd (Beijing, China) were at chromatographic grade purity.

Assay kits for total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were from the BioSino Bio-technology & Science Inc (Beijing, China). Rat Nitroreductase ELISA kit and Human Nitroreductase ELISA kit were obtained from the Beijing Luyuan Dade Biological Science and Technology Co., Ltd (Beijing, China).

Animals

Male Syrian golden hamsters (8-week old, 110-140 grams, the Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in the SPF-grade rooms and had free access to food and water.

Hamsters were randomly separated into two groups: the normal controls (26 animals) and hyperlipidemia models (26 animals). The animals fed with normal diet (ND) ad libitum served as controls and those fed with high-fat diet (HFD) for 6 weeks were the hyperlipidemia model hamsters 19.

The animal experiments were approved by the Laboratories' Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences (No.003541 and No.00000406). The research was conducted in accordance with the guidelines and ethics of the Chinese Council on Animal Care.

Instruments for detecting BBR and its metabolites

A liquid chromatography instrument coupled to an ion trap time-of-flight mass spectrometer (LC/MSn-IT-TOF) from the Shimadzu Corporation (Kyoto, Japan) was used to identify the chemical structures of BBR and its metabolites. Liquid chromatography with tandem mass spectrometry (LC-MS/MS 8050, Shimadzu Corporation, Kyoto, Japan) and gas chromatography with mass spectrometry (GC-MS QP2010, Shimadzu Cooperation, Kyoto, Japan) were used for the analysis and quantification of BBR and its metabolites in biological samples 14, 17, 20.

Blood lipid test

Blood lipids (TC, TG and LDL-C) were detected in the ND-fed (n=6) and HFD-fed hamsters (n=6) before and after BBR (100 mg/kg/day) oral administration on day 0, 7, and 10 using commercial enzymatic kits 19.

NR activity Determination

For NR activity of gut microbiota in hamsters, 1 gram colon contents were taken and transferred into a flask containing 20 mL anaerobic culture medium. After mixing, the cultures were incubated under anaerobic conditions with a N2 atmosphere at 37 °C for 24 hrs, followed by centrifugation at 5,000 rpm (15 min, 4 °C). The pellet was collected, washed twice in PBS and re-suspended in PBS at a weight/volume ratio of 1:500 (g/μL). Total bacterial protein was extracted via sonication disruptor (Scientiz-ⅡD; Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). NR activity of the extracted samples was measured with the rat or human ELISA kit. NR activity in human feces was tested using the method above.

For the NR activity in single strain bacteria, the intestinal bacteria Enterococcus faecium ATCC 35667 and Bacteroides fragilis ATCC 25285 (bought from the Guangdong Huankai Microbial Sci. & Tech. Co., Ltd, Guangdong, China) were cultured in an anaerobic condition without or with BBR (10 μg/mL, 24 hrs), respectively 17, 21. The NR activity was determined using the NR ELISA kit.

Bacterial composition analysis

Gut bacterial composition in the ND- or HFD-fed hamsters was analyzed via 16S rRNA genes analysis. The 16S rRNA genes were amplified using the specific primer for 16S V3-V4: 340F-805R to target the V3-V4 regions of 16S rRNA. PCR products were mixed in equimolar ratios. Subsequently, mixture of PCR products was purified with the GeneJET Gel Extraction Kit (QIAGEN, Germany). Sequencing libraries were generated using the NEXTflex Rapid DNA-seq kit for Illumina (New England Biolabs, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). Finally, the library was sequenced on the HiSeq2500 (Illumina, USA) platform and 250 bp paired-end reads were generated. Sequences were analyzed using the QIIME software package (Quantitative Insights Into Microbial Ecology). First, the reads were filtered by the QIIME quality filters. Next, we picked representative sequences for each OTU (operational taxonomic units) and used the RDP (ribosomal database project) classifier to annotate taxonomic information for each representative sequence. Sequences with a similarity over 97% were assigned to the same OTUs 22.

Clinical study

Forty individuals were randomly enrolled into the study in the Outpatient Section of the First Hospital of the Jilin University in Changchun in the spring of 2016. The study was approved by the institution ethnic committee of the hospital (clinical study No. 2016-270). Of the 40 individuals, 10 were healthy volunteers (Group 1; 7 males, 3 females; age 46.7 +/- 16.8) with blood lipids and glucose in normal range [blood cholesterol (mmol/L), 4.72 +/- 0.53; triglycerides (mmol/L), 1.09 +/- 0.33; LDL-C (mmol/L), 2.55 +/- 0.35; FBG (mmol/L), 4.90 +/- 0.51)], and 30 were patients (Group 2, 13 males, 17 females; age 58.8 +/- 13.7) with high level of blood lipids [blood cholesterol (mmol/L), 5.59 +/- 0.98; triglyceride (mmol/L) , 2.36 +/- 1.30; LDL-C (mmol/L) , 3.28 +/- 0.73; FBG (mmol/L), 6.56 +/- 2.97]. In the Group 2 individuals, 7 of them had both high blood lipids and glucose. The subjects were not in any drug treatment before enrollment.

Before BBR treatment, blood samples were taken for each individual for grouping purpose (the Group 1 and Group 2); fecal samples were also collected. The subject was orally treated with BBR (500 mg, once). Blood samples were collected 2 hrs after BBR oral administration, and placed at -20゜C. BBR plasma concentration was measured using LC-MS/MS as described 14, 17, 41. Before BBR treatment (in 24 hrs), 5 grams of fecal samples of the 32 individuals (9 healthy subjects and 23 patients) were collected and immediately prepared for an immediate measurement of NR activity.

Gut microbiota analysis of clinical individuals

Intestinal bacteria composition of healthy volunteers or patients with hyperlipemia / hyperglycemia was analyzed via 16S rRNA genes analysis. The 16S rRNA genes were amplified using the specific primer for 16S V3-V4: 338F-806R to target the V3-V4 regions of 16S rRNA. PCR products were mixed in equimolar ratios. Subsequently, mixture of PCR products was purified with the AxyPrep Gel Extraction Kit (Axygen Biosciences, USA). Sequencing libraries were generated using the NEBNext Ultr DNA Library Prep Kit for Illumina (New England Biolabs, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). Finally, the library was sequenced on the MiSeq (Illumina, USA) platform and 300 bp paired-end reads were generated. Sequences were analyzed using the QIIME software package (Quantitative Insights Into Microbial Ecology, V1.8.0). First, the reads were filtered by the QIIME quality filters. Next, we picked representative sequences for each OTU (Operational Taxonomic Units) and used the RDP classifier (Ribosomal Database Project) to annotate taxonomic information for each representative sequence via SILVA 119 ribosomal RNA (rRNA) database. Sequences with a similarity over 97% were assigned to the same OTUs.

Pharmacokinetics of BBR in hamsters and BBR concentration in human plasma

The ND-fed or HFD-fed hamsters were orally treated with BBR (100 mg/kg, n=6). Blood samples were collected from the posterior orbital venous plexus at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hrs. Hamsters were sacrificed at the last time point and brain, heart, kidney, lung and liver samples were collected. BBR was also given to the ND-fed (n=6) or HFD-fed (n=6) hamsters via intravenous injection (iv, 1 mg/kg). Additionally, small intestines were collected at 0.5 hrs after BBR oral administration (n=4). BBR, dhBBR and its metabolites were detected according to previously described methods 14, 17 and pharmacokinetic parameters were calculated by DAS 2.0.

For determining plasma BBR in hamsters treated with BBR (oral, 100 mg/kg/day, for 10 days, n=6), blood samples were collected on day-7 and day-10 0.5 hrs after treating with BBR. Blood BBR was quantified 14, 17, 41. Plasma (100 μL) was extracted with 1.25 mL ethyl ether after addition of 10 μL internal standard (250 ng/mL of palmatine) and 50 μL 0.5 M sodium hydroxide solution. Following centrifugation and separation, the organic phase (1 mL) was evaporated to dryness in a 40 ºC water bath with a sample concentrator (Nitrogen blowing instrument). The residue was reconstituted with 100 μL mobile phase (a mixture of acetonitrile and 0.5% formic acid with ratio of 1:4). An aliquot of 10 μL was injected into the LC-MS/MS 8050 system for analysis.

Statistical analysis

The statistical analyses were conducted using the two-way ANOVA and Student's t-test with the GraphPad Prism Version 5 (GraphPad Software, CA, USA). The data were expressed as means ± standard deviation. P values less than 0.05 were considered statistically significant.

Results

High-fat diet (HFD) changed gut microbiota composition and NR activity in hamsters

As shown in Figure , HFD treatment in hamsters for 10 days significantly increased blood cholesterol, triglycerides, and LDL-C (***P<0.001 for all) while simultaneously elevating NR activity in the HFD-hamster feces as compared to those fed with normal diet (**P<0.01). Composition analysis using microbiomic technique showed an increase in the NR-producing bacteria in the gut microbiota community (Figure ) with Bacteroides 23, Escherichia-Shigella 24, and Bifidobacterium 25 et al. showing the highest increase among the top 50 bacterial strains. These results suggested that HFD feeding altered the composition of gut microbiota. Also, incubating NR-producing bacteria Enterococcus faecium 26 and Bacteroides fragilis 23 with broth medium for 12 hrs elevated NR activity in the bacterial culture (Figure ). We thus concluded that the HFD or Broth treatment increased either the proportion of the NR-producers in the intestinal bacterial community and/or the NR activity in the bacteria.

Increased level of fecal NR promoted BBR oral bioavailability

NR is a group of bacterial enzymes that reduces BBR into dhBBR in intestine which has high intestinal absorption 17. Therefore, the increased activity of NR in the gut microbiota might help BBR to first enter into intestine wall tissue and then blood. As shown in Figure , both dhBBR and BBR were significantly higher in the intestinal tissue of the HFD-fed hamsters as compared to that in the hamsters fed with normal diet (*P<0.05, ***P<0.001, respectively). Consequently, determination of blood concentration of BBR showed that the C (peak concentration) of BBR in the HFD-fed hamsters was about 74.3% higher than that in those fed with normal chow (*P<0.05, Figure ). Also, the AUC (Area under the time concentration curve) measurement revealed a pattern similar to that of C (*P<0.05, Figure ). Accordingly, BBR content distributed in organs (liver, lung, kidney, heart, brain) of the HFD-hamsters was higher than that in the hamsters fed with normal diet with statistical significance seen in liver, lung and heart (Figure , *P<0.05 or **P<0.01). The distribution of BBR metabolites (M1-M5, Supplementary Figure ) in organs like intestine and liver revealed a pattern similar to that of BBR (Supplementary Figure ) confirming the results shown in Figure 2D. Although the BBR's AUC value in the HFD-fed hamsters increased by 66% over that in the normal diet controls after BBR oral administration (Figure ), difference between the HFD-fed hamsters and normal-diet hamsters in BBR's AUC value was not detectable if BBR was given by iv injection (1 mg/kg, Figure ) 27, demonstrating the critical role of gut microbiota in BBR absorption. The BBR iv injection dose was selected based on its bioavailability reported in animals 27. The oral bioavailability of BBR increased in the HFD-fed hamsters by 72% as compared to that in normal-diet group (Figure ). The results supported the role of intestinal NR in BBR absorption 17 and suggested that BBR might be more effective in individuals with high NR activity in intestine.

Figure 2

HFD treatment promotes BBR absorption in intestine and its oral bioavailability. (A) 0.5 hr after oral administration of BBR (100 mg/kg) both dhBBR and BBR were significantly increased in the small intestinal tissue of the HFD-fed hamsters as compared to that of the ND-fed hamsters (*P<0.05 for dhBBR, and ***P<0.001 for BBR, n=4 for both comparison); (B & C) C and AUC of BBR were much higher in the HFD-fed hamsters compared to those in the ND-fed hamsters 24 hrs after oral administration of BBR at 100 mg/kg (*P<0.05 for all; n=5 for the ND-fed group and n=6 for the HFD-fed group); (D) Increased levels of BBR in the main organs of the HFD-fed hamsters compared with that in the ND-fed hamsters 24 hrs after 100mg/kg oral administration of BBR (**P<0.01 and *P<0.05, n=5 for the ND-fed group and n=6 for the HFD-fed group); (E) Concentration curves of BBR in HFD-fed and ND-fed hamsters 24 hrs after oral administration of BBR (100 mg/kg) with a larger BBR AUC value in HFD-fed hamsters (n=5 for the ND-fed group, and n=6 for the HFD-fed group, respectively); (F) 24-hr concentration curves of BBR when given via iv route (1 mg/kg) in the ND-fed or HFD-fed hamsters both groups showing a similar pattern (n=4 for both); (G) BBR oral bioavailability increased in the HFD-fed hamsters by 72% as compared to that in the ND-fed hamsters (iv, 1 mg/kg; oral, 100 mg/kg).

BBR reduced blood lipids in the HFD-fed hamsters but not those fed with normal diet

We next extended the test into therapeutic efficacy. As shown in Figure (L, M & R), while BBR treatment did not alter the blood level of lipids in the hamsters fed with normal diet, its administration for 7 days (oral, 100 mg/kg/day) significantly decreased blood cholesterol, triglycerides and LDL-C in the HFD-fed hamsters (**P<0.01). Interestingly, along with the decrease in blood lipids from day 0 to day 10 (Figure , *P<0.05, **P<0.01), the NR activity in intestine also diminished gradually (Figure , **P<0.01). The NR level remained unchanged in the BBR-treated normal-diet hamsters (Figure ). It appeared that HFD feeding altered the composition of gut microbiota and caused high blood lipids. Simultaneously, the proportion of the NR-producing bacteria in gut microbiota as well as the NR activity in intestine increased (Figure ) which, in turn, promoted the conversion of BBR into dhBBR. Thus, there was an increase in oral bioavailability of BBR in the HFD-fed animals resulting in a good therapeutic outcome.

Blood level of BBR positively correlated with fecal NRs activity in clinical subjects

Our results in hamsters suggested that NR activity in gut microbiota might be a biomarker for BBR bioavailability in selecting patients for treatment. To test this hypothesis, 40 individuals were randomly enrolled into the study in the Outpatient Section of the First Hospital of the Jilin University in Changchun in the early spring of 2016 (Clinical approval number 2016-270). Out of the 40 individuals, 10 were healthy volunteers (Group 1), and 30 were patients (Group 2) with high level of blood lipids. As the first step of the trial, blood and fecal samples of the 40 subjects were acquired for blood lipids/glucose measurement and NR activity determination, respectively. BBR was given to the subjects orally followed by examination of blood BBR concentration 2 hrs later. As shown in Figure , individuals in Group 2 had a blood BBR level over 70% higher than that in normal subjects of Group 1 (*P<0.05). Considering the two groups together (n=40), the highest correlation was observed between blood BBR level and blood triglycerides concentration (Figure , r=0.83), suggesting that individuals with higher level of triglycerides might have better oral bioavailability of BBR. Furthermore, the hyperlipidemic patients in Group 2 had a significantly higher fecal NR activity level than that in the healthy individuals of Group 1 (Figure , **P<0.01) which was consistent with the observation in hamsters (Figure ). It appears that the elevated blood BBR level in Group 2 patients is the consequence of the increased activity of NR. The correlation analysis between fecal NR and blood BBR was also carried out in individuals from both Group 1 and Group 2. As shown in Figure , although the study size was relatively small (n=32; fecal samples of 8 individuals were not available in the study, with 1 in the Group 1 and 7 in Group 2), the fecal NR activity positively correlated with the blood concentration of BBR (r=0.70). The results demonstrated a significant role of NR in BBR absorption and suggested fecal NR activity as a potential biomarker for determining the therapeutic efficacy of BBR. Also, sex and age appeared to have no effect on the level of blood BBR in this study (Supplementary Figure ).

To examine the difference in gut flora between hyperlipidemic patients and healthy subjects, composition analysis for gut microbiota was performed by using human fecal samples randomly collected from the hyperlipidemic patients (n=3; labeled H-1, 2, 3) and healthy volunteers (n=3; labeled C-1, 2, 3). The results showed a significant difference in the gut microbiota composition between healthy and hyperlipidemic individuals (Figure ). As shown in Figure , Fusicatenibacter, Subdoligranulum, Enterobacter, Blautia and Lachnoclostridium were noticeably increased in the fecal samples of hyperlipidemic patients compared with the samples from normal volunteers. Although the NR information for some of these bacteria is not available, Enterobacter has been reported to be a NR-producer 28. Indeed, our previous results demonstrated that the Enterobacter cloacae of the gut microbiota efficiently converted BBR to dhBBR 17. Thus, the composition analysis of gut microbiota confirmed the difference of gut flora between healthy subjects and patients with hyperlipidemia which might contribute to the elevated level of NR in intestine.

Discussion

BBR is known to reduce blood lipids or glucose in patients with hyperlipidemia or type 2 diabetes, respectively 2-4. Our findings in the present study showed that BBR treatment lowered blood lipids and glucose effectively only in the HFD-fed hamsters with increased level of NR activity in the gut microbiota. On the other hand, BBR's lipid-lowering efficacy was not detectable in the normal diet-fed animals with the normal level of NR in gut microbiota. The results suggested that the intestinal NR activity could be at least part of mechanism for the lipid/glucose lowering activity of BBR. It appeared that HFD treatment resulted either in an increase in the fraction of NR-producing bacteria in gut microbiota and/or elevated the activity of NR in the bacteria. Consequently, the HFD treatment caused an enhanced BBR-to-dhBBR transformation in the intestine and therefore an increased oral bioavailability in vivo. Our clinical study also corroborated the findings in the hamster model and provided strong support for a positive correlation between the fecal NR activity and blood level of BBR, indicating a connection between BBR oral bioavailability and intestinal bacteria. This interesting correlation could be more significant if the size of study population is large. Composition analysis of the gut microbiota showed difference between the NR from healthy and hyperlipidemic individuals. Thus, we consider the fecal NR activity in gut microbiota to be a potential biomarker for the therapeutic efficacy of BBR.

The current concept for bioavailability of oral drugs is based on the evaluation of drug absorption through normal animal intestine 29. In principle, high oral bioavailability is considered a positive indication for drug therapeutic efficacy as it correlates with drug effectiveness 29. In the present study, we have shown that the oral bioavailability of BBR in the HFD-fed hamsters was much higher than that in the hamsters fed with normal diet (Figure ). This observation indicates that food composition might influence bioavailability of the oral drugs through gut microbiota and sheds new light on the existing knowledge of drug bioavailability when evaluated in appropriate animal models. For treating energy metabolic disorders, we consider the therapeutic selectivity of BBR, which is mediated through NR of gut microbiota, an unique advantage in clinical treatment, because less drug molecules enter the blood of healthy individuals or of those recovered from treatment (Figure ).

Therapeutic efficacy of drugs is known to be affected by genetic variations in drug targets or drug processing enzymes. A number of examples of genetic variations have been reported for drugs treating a variety of diseases such as hepatitis C infection 30, cancers 31, 32, blood coagulation et al. 33, representing a new frontier in drug therapy and personalized medicine 34, 35. For example, subtypes of CYP450s differ among races and individuals, resulting in variations in drug degradation rate 36, 37 and thus constituting an important factor in personalized drug therapy. In the present study we showed both in animals and clinical subjects that the individuals with high level of fecal NR activity had more blood BBR as compared to those with regular level of fecal NR, indicating a variation of oral bioavailability of BBR in human. It might cause different therapeutic efficacy in BBR treatment. To the best of our knowledge, this is the first report to show a functional molecule of gut microbiota acts as a biomarker for drug bioavailability and therapeutic efficacy.

Bacterial NR catalyzes the NADPH-dependent reduction of the nitro-group on nitroaromatic and nitroheterocyclic compounds as a mechanism of self-protection 38. The expression of bacterial NR genes seems to be governed by the MarRA and SoxRS regulatory mechanisms that are associated with bacterial response to antibiotics and chemicals in the environment 39-41. The NR genes are widely distributed in intestinal anaerobic bacteria 38 causing a steady up-take of BBR in human intestine (Figure ) 42. We show, both in the animal experiment and in the human study, that increasing about 10% of fecal NR activity in gut flora could raise blood BBR concentration by 65-70%, suggesting a highly efficient function of bacterial NR in transforming BBR into dhBBR. In our experiments, we used the NR activity assay to evaluate NR level instead of NR enzyme expression as, despite their high homology, sequence diversity of NR exists in different microorganisms 38. As fecal NR appears to be a sensitive enzyme for blood level of BBR, it might be a biomarker to predict the therapeutic efficacy of BBR in clinical practice, if verified in large scale clinical studies. Base on the finding in this investigation, we consider gut microbiota to be a new component in personalized medicine.

Supplementary figures.

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