Effects of hydroxypropyl methylcellulose on regulating effect of berberine hydrochloride intestinal microenvironment

Abstract: Objective: To investigate whether the combination of hydroxypropylmethylcellulose (HPMC) and berberine hydrochloride (BBR) will affect the regulation of intestinal microenvironment. Methods: C57BL/6 male mice were selected and divided into control group, HPMC (400 mg·kg-1) group and BBR (150 mg·kg-1) group. HPMC and BBR were physically mixed (H+B, 400 mg·kg-1) -1+150 mg·kg-1) group received intragastric administration for 3 consecutive weeks. The distal colon of the mice was collected, and the histopathological morphology of the colon was detected by hematoxylin-eosin (HE) staining; 16S rRNA gene sequencing was used to explore the changes in the structure of the intestinal flora of the mice. Results: Compared with the control group, the Alpha diversity of the intestinal flora of mice in the BBR group was significantly reduced, and the results of Beta diversity showed that the composition of the flora was significantly changed. Proteobacteria, Verrucomicrobia and Ekman The relative abundance of Akkermansia increased significantly. However, the physical mixed administration of HPMC and BBR can further promote the decrease of Alpha diversity of intestinal flora in mice caused by BBR, and enhance its antibacterial effect; in terms of Beta diversity, the intestinal flora of H+B group and BBR group Group composition produced differences while significantly enhancing BBR's enrichment of Proteobacteria, Verrucobacteria, and Ekmania. Conclusion: The physical mixing of HPMC and BBR can positively promote the flora regulation of BBR, presumably because the high viscosity can enhance the adhesion of berberine on the intestinal tract, so that berberine can fully contact the intestinal flora.

Key words:hydroxypropyl methylcellulose; berberine hydrochloride; intestinal flora; intestinal microenvironment

Berberine hydrochloride (BBR) is an alkaloid mainly isolated from Coptidis Rhizome and Phellodendron barkberry, which has multiple biological functions. BBR has low polarity, low solubility, poor permeability, extremely low blood concentration, and low bioavailability, but it is effective in the treatment of gastrointestinal inflammation, metabolic related diseases (such as type 2 diabetes), Alzheimer's disease and Parkinson's disease etc. have significant medicinal effects. According to the traditional pharmacological concept, the drug needs to enter the blood and reach a certain concentration of active ingredients to exert its therapeutic effect. This theory cannot fully explain the various pharmacological activities of BBR. Current research points out that besides a small amount of absorption into the blood, another key reason for BBR to exert the above-mentioned medicinal effects may be the regulation of flora brought about by its poor absorption and large accumulation in the intestinal tract, such as the increase of short-chain fatty acids (especially is the relative abundance of butyric acid-producing bacteria, etc. Therefore, giving full play to the regulation of BBR's flora may be a key means to improve its efficacy.

As more and more research focuses on intestinal health, the black box of the relationship between intestinal flora and the outside world and the body is gradually being opened. Studies have found that the stability of intestinal microecology and daily eating habits will have an impact on intestinal function. Multifaceted effects such as intestinal permeability and intestinal barrier function. Studies have shown that intestinal permeability is related to many diseases, and paying attention to intestinal permeability is very important for disease prevention and treatment. According to reports, mild damage to the intestinal barrier can cause weak intestinal inflammation, increased expression of pro-inflammatory cytokines, increased susceptibility of the body, and susceptibility to adhesion and invasive Escherichia coli infection, resulting in intestinal disorders Further aggravation, intestinal permeability increases, harmful small molecules enter the body, and aggravate body inflammation. Among them, the initial manifestation of impaired intestinal barrier function is the thinning of the intestinal mucus layer, which causes damage to the structure of the mucosal layer, and the probability of cysts in the intestinal epithelium increases.

As a pharmaceutical excipient, hydroxypropyl methylcellulose (HPMC) is recorded in the 2020 edition of the Pharmacopoeia of the People's Republic of China. It has many applications in the biomedical industry, mainly for coating materials, film materials and sustained-release preparations Controlled release materials, such as adhesive materials in tablets, coating materials for improved aspirin enteric-coated tablets, and skeleton materials for drug sustained-release tablets. On the one hand, as one of the most commonly used hydrophilic carriers, HPMC is widely favored in formulation research. Studies have shown that in the oral mucosal drug delivery system, HPMC can enhance the ability of the drug to adhere to the oral mucosa, thereby increasing the contact time of the drug with the oral mucosa to better exert the drug effect. Another study showed that, as one of the matrices of in situ gels, as the concentration of HPMCF4M increased, the viscosity of the gels increased, and the degree of delayed drug release also increased accordingly. On the other hand, as a non-fermentable dietary fiber, HPMC is considered as a potential prebiotic fiber, and it has been reported that it can regulate the alpha diversity and composition of intestinal flora in high-fat-fed mice.

At present, the functional cognition of pharmaceutical excipients is gradually deepening, and the standard for selecting pharmaceutical excipients in the traditional sense is constantly being refreshed. It is no longer limited to selecting excipients with high safety, good biocompatibility, and no interaction with active substances. Its interaction with intestinal flora has also been gradually taken into consideration. Studies have reported that various pharmaceutical excipients can affect the health of the body and the efficacy of drug treatment by regulating the flora. The influence of the role should also be given full attention. Similar to BBR, HPMC is not easy to be metabolized in the human body, and at the same time has high viscosity, which can make BBR stay in the intestinal tract, which creates a more favorable environment for the drug to fully contact the intestinal flora so as to better play a role in regulating the flora. conditions of. In this study, HPMC was taken as an example to explore the regulation effect on the intestinal microenvironment when it was physically mixed with BBR. The research on group regulation affecting the efficacy of BBR provides a reference.

1. Materials

1.1 Experimental animals

40 specific pathogen-free (SPF) grade male C57BL/6 mice, weighing 18-20 g, were purchased from Speiford (Beijing) Biotechnology Co., Ltd., the production license number is SCXK (Beijing) 2016-0006, and were bred In the SPF-level animal room of the Institute of Medicinal Plants, Peking Union Medical College, the mice were reared under constant temperature and humidity conditions, the ambient temperature was (22±2) °C, the relative humidity was (50±5) %, and the light was 12 h/12 h. Alternate dark, normal feed and purified water. The animal experiments involved in this study were handled in accordance with the animal ethics procedures and norms of Peking Union Medical College, and the approval number is SLXD-20201221013.

1. 2 Instruments

Secura224-1CN electronic analytical balance (Germany Satrorius company); KQ-500DE numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); SP131010-33Q heating magnetic stirrer (US Barnstead Thermolyne company); L550 desktop low-speed large Capacity centrifuge (Eppendorf, Germany); TL-48R crushing and grinding instrument (Shanghai Wanbai Biotechnology Co., Ltd.); DYY-6C electrophoresis instrument (Beijing Liuyi Instrument Factory); NanoDrop 2000 ultra-micro spectrophotometer ( Thermo Fisher, USA); GeneAmp®9700 polymerase chain reaction (PCR) instrument (ABI, USA); Quantus™ miniature fluorometer (Promega, USA); Miseq PE300/NovaSeq PE250 platform (Illumina, USA); Aperio CS2 compact desktop slide scanner (Leica Microsystems, Germany).

1.3 Reagent

HPMC F4M (lot number: D180I8Q012, KIMA CHEMICAL CO.,LTD); BBR (lot number: ZLSC2020072617, purity >98%, Nanjing Zelang Co., Ltd.); E. Z. N. A. ® soil DNA kit (Omega Bio-Tek, USA); AxyPrep DNAGel Extraction Kit (Axygen, USA); Agarose (Biowest, Spain); Quantus™ Fluorometer (Promega, USA); NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, USA); Hematoxylin-eosin (HE) staining kit ( Beijing Suolaibao Technology Co., Ltd.); primers were synthesized by Beijing Yikebaide Biotechnology Co., Ltd.

2. Method

2.1 Solution preparation

HPMC aqueous solution (40 mg·mL-1) was prepared and magnetically stirred at room temperature for 24 h, and it was swollen to a clear, transparent viscous liquid before use; BBR aqueous solution (15 mg mL-1) was prepared and ultrasonicated for 30 min (100 W, 40 kHz) to obtain a suspension. Prepare the aqueous matrix of HPMC first, and then add BBR into the matrix to obtain a mixed solution with a mass concentration of HPMC of 40 mg·mL-1 and a mass concentration of BBR of 15 mg·mL-1. use.

2.2 Grouping and administration

Twenty-four C57BL/6 mice were fed adaptively for 1 week, and then randomly divided into control group, HPMC (400 mg·kg-1) group, BBR (150 mg·kg-1) group, HPMC and BBR group Physical mixed (H+B, 400 mg·kg-1+150 mg·kg-1) group, 6 rats in each group. Oral administration, once a day, mice in the control group were given distilled water by intragastric administration, for a total of 3 weeks. Among them, the dosage of BBR is obtained from literature research, and the middle value in the commonly used dosage range of BBR is selected for exploration. The dosage of HPMC is the maximum dosage that can be administered orally after screening in the preliminary experiment.

2.3 Pathological examination of the colon

Mice were killed by cervical dislocation, the distal colon tissues were removed, the contents were removed, fixed with 4% tissue cell fixative solution, embedded in paraffin, eluted with gradient volume fraction alcohol, and cut into 3.5 μm thick sections according to standard procedures. Thin sections were stained with HE, and three fields of view were randomly selected from each section for observation under an optical microscope.

2.4 High-throughput sequencing analysis of intestinal flora

After 3 weeks of drug intervention, the fresh feces of the mice in each group were collected, placed in cryopreservation tubes, quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator for future use. The mouse feces were taken, and the total DNA of the microbial community was extracted using the E. Z. N. A. ® soil DNA kit. The quality of the DNA extraction was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined. 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3~V4 variable region of the 16S rRNA gene by PCR. After the PCR products of the same sample were mixed, the PCR products were recovered by 2% agarose gel, purified by AxyPrep DNA Gel Extraction Kit, detected by 2% agarose gel electrophoresis, and quantified by Quantus™ Fluorometer. The NEXTFLEX Rapid DNA-Seq Kit was used for library construction, and the Miseq PE300/NovaSeq PE250 platform was used for sequencing.

2.5 Statistics

Due to the accidental death of one mouse in the H+B group, the number of samples in each group involved in the statistics is 6 in the control group, 6 in the HPMC group, 6 in the BBR group, and 5 in the H+B group. Colon tissue section observation and thickness marking were analyzed using CaseViewer software. Operational taxonomic units (OTUs) were plotted using R 3.2.0 software and GraphPadPrism software. The Alpha diversity analysis of the flora used ObservedOTUs, and the Chao1 richness index, Shannon diversity index, Simpson diversity index, and Beta diversity analysis used non-metric multidimensional scaling (NMDS) analysis. Data analysis was performed by one-way analysis of variance (ANOVA) and nonparametric Tukey test. P<0.05 was regarded as a statistically significant difference between groups.

3. Results and Discussion

3.1 Effects of HPMC and BBR on mouse colon tissue

The results of HE staining of colonic tissue showed that the mucosal structure of the colonic tissue of the mice in each group was intact, the intestinal villi had no obvious defects, and no obvious edema was observed, indicating that the mice in each group did not have obvious inflammatory reactions.

The thickness of the mucosal layer, submucosa and sarcolemma (the sum of the thickness of the serosal layer and the muscular layer) of the mouse colon tissues in each group was analyzed. The results showed that after 3 weeks of intervention, the thickness of the more critical colonic mucosal layer in each group No significant changes occurred. From the results of HE staining of colon tissue and the analysis of the thickness of each part of intestinal tissue, it can be concluded that the intervention of HPMC and BBR has no significant effect on the mechanical barrier of the colon.

3.2 Effects of HPMC and BBR on the diversity and composition of intestinal flora in mice

3. 2. 1 Overlap of sample OTUs

The Venn diagram was used to analyze the unique OTUs and common OTUs in the intestinal flora of mice in each group. The results showed that the OTUs in the control group, HPMC group, BBR group, and H+B group were 2026, 2095, 299, and 161, respectively. There were 323 unique OTUs in the HPMC group, more than those in the control group (267), BBR group (96), and H+B group (7). The HPMC group shared 1577 OUTs with the control group, the BBR group shared 7 OUTs with the control group, and the BBR group shared 22 OUTs with the H+B group. It shows that in terms of the number of OTUs and unique components, the HPMC group is more similar to the control group, and the BBR group is more similar to the H+B group.

3. 2. 2 Alpha Diversity

Alpha diversity analysis was performed on the OTUs sequences obtained by sequencing the intestinal flora of mice in each group. The number of species observed in the BBR group and the H+B group was significantly lower than that in the control group and the HPMC group (P<0. 001). The Chao1, Shannon, and Simpson indices calculated from the number of OTUs were analyzed. Compared with the control group, the Shannon index did not change significantly in the HPMC group except that the uniformity within the group decreased; the uniformity within the group in the BBR group was better, and the Shannon index decreased significantly (P<0.001). Compared with the BBR group, the Shannon index of the H+B group was further reduced, and the difference was statistically significant (P<0.05), and the Simpson index was significantly lower, and the difference was statistically significant (P<0.05), that is, compared with HPMC Combined use enhanced the antibacterial ability of BBR. It is speculated that this is related to the physical properties of HPMC, such as metabolic inertia, high viscosity, enzyme inhibition and colloidal protection, etc. Among them, the property of enzyme inhibition can play a role in maintaining viscosity stability, so that HPMC can maintain excellent viscosity in different environments. After a long period of physical mixing, HPMC fully swells to make BBR evenly dispersed and prevents it from forming a precipitate at the bottom of the solution. After contacting with water, HPMC can form a transparent and flexible film.

BBR is wrapped and protected when it passes through the stomach and small intestine. When it reaches the colon, the physical mixing system of HPMC and BBR makes BBR more uniform in contact with the intestinal flora, thus promoting the tendency of BBR to reduce the Alpha diversity of intestinal flora.

3. 2. 3 Beta Diversity

The NMDS diagram was drawn to analyze the degree of difference between the control group, HPMC group, BBR group and H+B group, and Beta diversity analysis was performed. The greater the degree of separation between different groups, the more obvious the difference between groups. The results showed that the distance between the HPMC group and the control group was relatively close; the distances between the BBR group and the H+B group were far from the control group, and the difference was large; the distance between the BBR group and the H+B group was relatively small, and the difference was small. The flora composition of the BBR group and the H+B group was significantly different from the other two groups, and the BBR intervention effectively played a role in regulating the flora.

3.2.4 Composition of gut flora at the phylum level

At the phylum level, the dominant flora in the composition of the intestinal flora of mice in each group included Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Verrucobacteria. Bacteria (Verrucomicrobia). The relative abundance of Bacteroidetes in the control group was 57.11%, the BBR group was 43.75%, and the H+B group was 35.48%; the relative abundance of the control group Firmicutes was 33.57%, and the BBR group was 35.48%. group was 24. 22%, H+B group was 16. 85%; the relative abundance of actinomycetes in the control group was 3. 67%, and the BBR group was 0. 01% (P<0. 05), H+ Group B was 0. 01% (P<0. 05). In contrast, BBR intervention significantly enriched Proteobacteria and Verrucomicrobia. The relative abundance of Proteobacteria in the control group was 2. 00%, 11. 99% in the BBR group (P<0. 001), and 14. 65% in the H+B group (P<0. 001); The relative abundance of Microbacteria was 2. 78%, the BBR group was 19. 63% (P<0. 05), the H+B group was 33. 00% (P<0. 001), and the H+B group was rich in The ability to gather Verrucomicrobia was better than that of BBR group. Among the changes of these dominant bacterial phyla, the increase of Verrucomicrobial phylum was the most prominent, which was speculated to be closely related to the substantial increase in the relative abundance of Akkermansia belonging to Verrucomicrobial phylum.

3.2.5 Composition of intestinal flora at the genus level

At the genus level, the relative abundance of Lachnospiraceae in the control group was 1. 219 0%, that in the BBR group was 0. 003 0% (P<0. 05), and that in the H+B group was 0. 0006% (P<0. 05); the relative abundance of Ruminococcaceae_UCG-014 in the control group was 1. 430%, 0. 013% in the BBR group, and 0 in the H+B group; ) was 1. 319%, the BBR group was 0. 074% (P<0. 05), and the H+B group was 0; the total relative abundance of other unidentified genera in the control group was 70. 92%, BBR was 34. 73% (P<0. 001), H+B group was 12. 88% (P<0. 001); compared with BBR group, H+B group was significantly lower (P<0. 05) . For the above genera, there was no significant change in the HPMC group, and BBR caused a decline in the relative abundance to varying degrees. The H+B group and the BBR group had the same trend. Except for the unidentified genus, the variation range of other genera was small, no more than 2%.

BBR can enrich some genera, such as Blautia, Ekmansia and Escherichia-Shigella. The relative abundance of Blautia in the control group was 1. 525%, the BBR was 12. 720% (P<0. 001), and the H+B group was 8. 717% (P<0. 05). Blautia is the main butyric acid-producing bacteria, which belongs to short-chain fatty acid-producing bacteria. The production of short-chain fatty acids can promote the formation of intestinal tight junction proteins in the body and the growth of intestinal beneficial bacteria, which can alleviate the pathogenesis of pathogenic factors. The immune inflammation caused by the increased intestinal permeability played a good role in repairing and protecting the intestinal barrier function; the relative abundance of Ekkermansia in the control group was 2. 776%, and that in the BBR group was 19. 630% ( P<0. 05), H+B group was 33. 000% (P<0. 001), which enhanced the effect of BBR on enriching Ekmansia. As a star genus, Ekmania belongs to Verrucomicrobia, and BBR significantly up-regulated the abundance of Verrucomicrobia and Ekmania, which is consistent with previous findings that Ekmania is The main genera of mucus-colonizing microorganisms are closely related to the protective effect of the mucus layer on intestinal tissues. Studies have shown that the relative abundance of Ekkermansia in the intestines of patients with Crohn's disease and ulcerative colitis decreases, which is negatively correlated with the disease state. Therefore, the enrichment of this bacterium by BBR may be the key reason for its restoration of the thickness of the mucus layer. The relative abundance of Escherichia-Shigella in the control group was 0. 002%, that in the BBR group was 10. 150% (P<0. 001), and that in the H+B group was 11. 890% (P<0. 001). For each of the above genera, HPMC intervention did not make the difference in the relative abundance of the bacteria statistically significant. It is worth noting that the relative abundance of Parasutterella in the control group was 0. 961%, and that in the HPMC group was 0. 116% (P<0. 001). Compared with the control group, Parasutterella in the BBR group 441%。 The relative abundance of Sartreella did not change significantly, while the H+B group had a slight downward trend, and the relative abundance of Sartreella was 0. 441%. Parasartreus is involved in the maintenance of bile acid homeostasis metabolism, and its homeostasis imbalance may have adverse effects on the intestinal barrier. The relative abundance of this bacterium in the HPMC group decreased slightly, and it returned to normal after being combined with BBR, avoiding the intestinal barrier. Road microecological disorder.

Taken together, whether it is from the change of Alpha diversity or the composition of intestinal flora, the regulation of BBR's flora has been positively promoted after being physically mixed with HPMC. Due to the high viscosity of HPMC, the viscosity of HPMC F4M selected in this study can reach 4000 mPa·s at a concentration of 2%. In this study, 4% HPMC was selected. Studies have pointed out that HPMC F4M can make drugs better adhere to the oral mucosa. It is speculated that after 4% of the high-viscosity HPMC is physically mixed with BBR, BBR can fully adhere to the intestinal mucosa, increasing the relationship between BBR and colonization in intestinal mucus. The probability and time of the contact of the intestinal flora on the upper layer directly promote the regulation of the flora of BBR. At the same time, studies have shown that HPMC F4M can delay the release of drugs, speculating that it can increase the retention of BBR in the intestine, and to some extent also promote its interaction with intestinal flora. On the other hand, from the perspective of BBR absorbing into the blood to play a role, since HPMC is a kind of hydrophilic polymer, when it is physically mixed with BBR with low solubility, there is a possibility to increase the solubility of BBR, thereby promoting the entry of BBR into the blood Play a medicinal effect.

4 Conclusion

In this study, the combined use of BBR and HPMC and the possible changes in the intestinal flora and intestinal microecology of mice caused by the intervention of BBR alone were carried out. After being physically mixed with BBR, it can positively promote the function of BBR itself, and can enhance the broad-spectrum antibacterial ability of BBR, and at the same time enhance the effect of BBR on enriching Ekmansia. It is speculated that this is enhanced when HPMC is physically mixed with BBR It adheres to the intestinal mucosa and delays the release of BBR, thereby promoting the full contact between BBR and intestinal flora to better play the role of flora regulation. In addition, the difference in the effects of other types of HPMC such as E4M and K4M with the same viscosity but different degrees of substitution of methoxy and hydroxypropyl groups on BBR’s intestinal microecological regulation can also be considered, so as to explore mechanism of action. In addition, pectin, carbomer, sodium alginate and other pharmaceutical excipients that can be used as gel matrix also have the potential to affect the regulation of BBR intestinal microecology. Considering the selection criteria of pharmaceutical excipients in formulation research.