Application of artesunate in improving ulcerative colitis through Alistipe-AMPK axis

Abstract

The invention discloses application of artesunate to improvement of ulcerative colitis through an Alistipe-AMPK axis, relates to the technical field of biological medicines, finds that the artesunate can regulate intestinal flora for the first time, and preliminarily explores a special anti-inflammatory action mechanism of the artesunate in UC (ulcerative colitis) relief. It is proposed for the first time that the Alistipe-AMPK axis can play a role in UC attack and treatment, and a new thought is provided for UC treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to application of artesunate in improving ulcerative colitis via the Alistipes-AMPK axis. Background Art

[0002] Inflammatory bowel disease is a group of intestinal diseases characterized by chronic nonspecific inflammation of the ileum, colon, and rectum. The main clinical subtypes include Crohn's disease (CD) and ulcerative colitis (UC). Epidemiological studies have shown that the incidence of UC is significantly higher than that of CD. Its lesions have a continuous distribution characteristic, typically involving the rectum and extending to the proximal colon. The clinical triad is characterized by abdominal pain, mucopurulent and bloody stools, and tenesmus. Endoscopy shows diffuse mucosal congestion, erosion, and pseudopolyp formation. The characteristic pathological manifestations are crypt abscesses and decreased goblet cells.

[0003] Artemisinin, extracted from Artemisia annua by Tu Youyou's team, has been successfully used for the treatment of malaria. Its derivative artesunate (ARS) also shows a wide range of activities. Studies have found that ARS can alleviate the symptoms of experimental enteritis mice, inhibit colon damage, regulate inflammatory factors, enhance intestinal barrier function, and reduce cell apoptosis and oxidative stress. These effects can be achieved through multiple mechanisms, including regulating the NF-B signaling pathway, inducing apoptosis of specific cells, inhibiting endoplasmic reticulum stress and Th1 / Th17 response. However, the therapeutic potential of artesunate in UC has not been fully explored. Therefore, the application of artesunate to improve ulcerative colitis through the Alistipes-AMPK axis is proposed to solve the problems raised above. Summary of the invention

[0004] The purpose of the present invention is to provide an application of artesunate to improve ulcerative colitis through the Alistipes-AMPK axis to solve the problems in the current market raised by the above background technology. The present invention studies the flora regulation effect of artesunate and explores its special anti-inflammatory mechanism in UC relief around intestinal flora.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Artesunate improves ulcerative colitis via the Alistipes-AMPK axis.

[0007] Preferably, the Alistipes species is Alistipes finegoldii strain.

[0008] A pharmaceutical composition for treating ulcerative colitis comprises artesunate, at least one probiotic of the genus Alistipes, and an AMPK pathway activator.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention uses a DSS-induced mouse acute enteritis model, and through methods such as DAI evaluation, HE, IHC, IF, RT-qPCR, WB, 16S rDNA, reference RNA seq transcriptome sequencing, and omics combined analysis, it is finally proved that artesunate can effectively improve experimental mouse acute enteritis, and its anti-inflammatory mechanism is achieved through the regulation of the flora, affecting the target functional bacteria Alistipes, and then promoting the expression of the AMPK / ACC pathway.

[0011] Specifically, it can be seen from the present invention that artesunate can significantly improve the disease activity, colon shortening, colon histological pathological damage, secretion of intestinal epithelial goblet cell mucin MUC2 and continuity of tight junction protein ZO-1 in DSS-induced enteritis mice, and reduce the mRNA content of proinflammatory factors IL-1β, IL-6, and TNF-α. Artesunate can also restore the species diversity, richness, uniformity and flora structure of the intestinal flora of experimental enteritis mice, promote the growth of potential beneficial bacteria (such as Firmicutes, Patescibacteria, Ruminococcaceae_UCG-014, Alistipes, etc.), and inhibit the colonization of conditional pathogens (such as Proteobacteria, Bacteroidetes, Escherichia coli-Shigella, Erysipelatoclostridium, etc.).

[0012] In the present invention, the joint omics analysis found that the target Alistipes genus is related to the target AMPK signaling pathway, that is, artesunate can affect the AMPK signaling pathway by regulating the Alistipes genus, thereby exerting an anti-inflammatory effect. Artesunate and Alistipes finegoldii intervention can both upregulate the mRNA and protein expression levels of genes related to the AMPK / ACC pathway, suggesting that the AMPK / ACC pathway may be one of the key mechanisms for artesunate to exert its anti-inflammatory effect.

[0013] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The figure shows the effect of artesunate of the present invention on the DAI score (A), colon length (B), general appearance of the colon (C), histopathological score (D), and colon pathology (E) score of DSS-induced enteritis mice; among which, (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0015] Figure 2 The figure shows the effect of artesunate of the present invention on the expression of MUC2 (A), ZO-1 (B) and inflammatory factor levels (C) in the colon of mice with DSS-induced enteritis; wherein, (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0016] Figure 3 The figure shows the effect of artesunate on the α diversity and β diversity of intestinal flora in mice with DSS-induced enteritis; (A): Venn diagram of OTU (α diversity); (B): Shannon index (α diversity); (C): UPGMA tree (β diversity); (D): PCoA analysis (β diversity); (E): relative abundance of phylum; (F): relative abundance within genus. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0017] Figure 4 The figure is a graph showing the effect of artesunate of the present invention on the indicator species of intestinal flora in mice with DSS-induced enteritis; wherein: the index analysis in the genus (A); the index analysis in the species (B); and the LEfSe analysis (C).

[0018] Figure 5 This is a heat map of the Pearson correlation analysis of artesunate in the present invention on the colon tissue of DSS-induced enteritis mice; wherein, (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0019] Figure 6 Figure 2 shows the DAI score (A), colon length and general appearance of the colon (B), colon pathology and histopathology score (C) of Alistipes finegoldii in mice with DSS-induced enteritis. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Embodiment 1

[0022] For reagents, all chemicals were of the highest purity. Dextran sulfate sodium (0216011080) was purchased from MPBiomedicals (USA). Corn oil (HY-Y1888), artesunate (HY-N0193) were purchased from Med Chem Express (USA). Prime Script RT Reagent Kit with DNA Erase and TB Green Premide were from Takara (Japan). PCR primers were from Sangon Biotech (China). Primary antibodies against MUC2 (Ab-272692), ZO-1 (Ab-221547), AMPK (Ab-32047) and p-AMPK (Ab-133448) Abcam (UK). Primary antibodies against ACC (3676) and p-ACC (1-1818) Cell Signaling Technology (USA). Alistipes finegoldii (BNCC354337) was purchased from BeNa Culture Collection (China). Gifu anaerobic medium (HB8518-1) was purchased from Hopebio (China).

[0023] For the animal model, male BALB / c mice from the Guangdong Medical Experimental Animal Center (certificate number: SYXK 2013-0002) were selected, with an average weight of 18-22 g and an age of 6-8 weeks. The mice were placed in a special pathogen-free (SPF) level environment at 24°C ± 1°C, with a light / dark cycle of 12 h, and a relative humidity of 50-70%. The mice had free access to food and water during feeding, and the experiment began after one week of adaptive feeding. All animal experiments have been approved by the Animal Ethics Committee of Guangzhou First People's Hospital.

[0024] The first batch of 30 mice were randomly divided into the following 5 groups: healthy control group (Control), enteritis model group (DSS), low-dose artesunate intervention group (DSS+L-ARS), medium-dose artesunate intervention group (DSS+M-ARS) and high-dose artesunate intervention group (DSS+H-ARS), with 6 mice in each group.

[0025] The second batch of 30 mice were randomly divided into three groups: healthy control group (Control), enteritis model group (DSS), and Alistipes finegoldii intervention group (DSS+AF), with 10 mice in each group.

[0026] The mouse acute enteritis model was established by adding 2.5% DSS to the sterile drinking water of mice for 7 days.

[0027] The enteritis model group, low / medium / high dose artesunate intervention group, and Alistipes finegoldii intervention group had free access to sterilized drinking water containing 2.5% DSS, and the healthy control group only received normal sterilized drinking water throughout the experiment. The mice in the Control group and DSS group were given 200 μL sterile PBS solution by gavage every day, the mice in the DSS+L / M / H-ARS group were given artesunate (20, 40, 80 mg / kg / mouse) dissolved in 200 μL corn oil, and the mice in the DSS+AF group were given fresh Alistipes finegoldii (108-9 CFU / mL / mouse) dissolved in 200 μL sterile PBS solution.

[0028] During the whole experiment, the weight, stool consistency and blood in the anus or stool of the mice were checked every day, and the DAI was calculated according to (weight loss score + stool quality score + blood in stool score) / 3 and statistical analysis was performed. After the experiment, fresh colon tissues of mice in each group were taken from the anus about 1.0 cm and fixed in 4% paraformaldehyde overnight, routinely dehydrated and embedded, 4 μm sliced ​​and stained with hematoxylin-eosin. Colon histopathological evaluation was performed according to the standard.

[0029] During the whole experiment, Figure 1 As shown in the results, the mice in the healthy control group were generally in good condition. The mice in the enteritis model (DSS) group began to show symptoms of colitis such as significant weight loss, unformed feces, and bloody stools on the third day after being given sterile drinking water containing 2.5% DSS. The DAI at the end of the experiment was significantly increased (P=0.0006), and the colon length was significantly shortened (P<0.001). The mice in the low / medium / high dose artesunate intervention groups were in better condition than those in the DSS group, and the DAI at the end of the experiment was significantly reduced (P=0.0986; P=0.0216; P=0.0089), and the degree of colon shortening was significantly improved (P=0.0487; P=0.0161; P=0.0024). Pathological staining showed that the mice in the DSS group had a decrease in goblet cells in the colon tissue, loss of crypt structure, submucosal edema, and inflammatory cell infiltration compared with the mice in the Control group. The above manifestations were significantly improved in the mice in the DSS+L / M / H-ARS group. The pathological histological scores showed that except for the low-dose group, which had a significant trend but no statistical difference (P=0.0629), the medium and high-dose groups were all statistically significant (P=0.0013; P<0.0001).

[0030] In summary, artesunate has the best therapeutic effect at a dose of 40 mg / kg / d and above, and there is no obvious drug dose dependence. The DSS+M-ARS (40 mg / kg / d) group was used for subsequent observation and analysis.

[0031] Intestinal mucosal epithelial cells and their tight junctions and intestinal mucus layer can effectively resist pathogens, toxins and other substances in the intestinal cavity as a mechanical barrier, and play an important role in maintaining the normal structure of the intestine. The expression of MUC2 in the goblet cell mucin of mice and the continuity of intestinal epithelial tight junction proteins ZO-1 and Occludin were evaluated by immunohistochemical staining and immunofluorescence. The results showed that compared with the control group, DSS stimulation significantly inhibited the secretion of MUC2 and the expression of ZO-1, while ARS intervention significantly restored the decreased secretion of MUC2 in the goblet cells of the colon of DSS-induced enteritis mice (P=0.0092; P=0.0132) and the continuity of ZO-1.

[0032] In addition, the level of inflammatory factor secretion is a commonly used biological marker for evaluating acute and chronic colitis. Figure 2 As shown in the figure, RT-qPCR analysis showed that the relative expression levels of proinflammatory factors IL-1β, IL-6, and TNF-α mRNA in the colon tissue of mice with DSS-induced enteritis were significantly higher than those in the Control group (P=0.0015; P=0.0412; P=0.0065), while the intervention of ARS inhibited the increase in the relative expression levels of proinflammatory factor mRNA (P=0.0244; P=0.0448; P=0.0329).

[0033] Embodiment 2

[0034] Paraffin-embedded tissue sections were dewaxed and antigen retrieval was performed according to standard experimental procedures.

[0035] For IHC: The sections were treated with 3% hydrogen peroxide solution in methanol for 30 min, then blocked with 10% goat serum for 60 min, and diluted primary antibody MUC2 (Abcam, USA) was added and incubated overnight at 4°C. The next day, diluted secondary antibody was used for incubation, DAB staining, and hematoxylin counterstaining of cell nuclei were performed, and finally microscopic imaging analysis was performed. The sections were read blindly by two people according to the standard.

[0036] For IF: The sections were blocked with 10% goat serum for 60 min, and then diluted primary antibody ZO-1 (Abcam, USA) was added and incubated overnight at 4°C. The next day, diluted secondary antibody was used for incubation, and anti-fading mounting medium containing DAPI was added. The slides were left to dry at room temperature, and finally microscopic imaging analysis was performed, and light was protected throughout the process.

[0037] Total RNA was extracted from tissues using the Trizol method and amplified using the following primers:

[0038]

[0039] Table 1 Primer sequence information

[0040] Perform western blot analysis:

[0041] Colon tissue samples were lysed in RIPA (Thermo Fisher Scientific, USA) buffer containing PMSF (Thermo Fisher Scientific, USA). Total protein content was determined using the BCA method. Proteins were separated on SDS-PAGE gels and transferred to PVDF membranes. The cell membranes were blocked with 5% skim milk and incubated overnight at 4°C using primary antibodies p-NFκB / NFκB (CST, China), p-AMPK / AMPK (Abcam, USA), and p-ACC / ACC (CST, China). The membranes were incubated with HRP-conjugated secondary antibodies the next day. ECL detection and image acquisition analysis were used.

[0042] Perform 16S rDNA high-throughput sequencing:

[0043] The DNA of microorganisms was extracted using the HiPure Stool DNA extraction kit according to the instructions. The extracted DNA was quality tested using a NANODROP micro-spectrophotometer and agarose gel electrophoresis. The forward primer (5′-CCTACGGGNGGCWGCAG-3′) and the reverse primer (5′-GGACTACHVGGGTATCTAAT-3′) were used for PCR amplification of the V3-V4 fragment. Finally, the AxyPrep DNA gel extraction kit was used for purification and quantification was performed using the ABI StepOnePlus real-time PCR system. The purified amplicons were double-end sequenced on the platform according to standard operations. OTU represents the sequence or ASV sequence alignment to the SILVA database, and the species classification annotation was performed using the naive Bayesian model of the RDP annotation software (technical support provided by Guangzhou Kidio Biotechnology Co., Ltd.).

[0044] Perform referenced RNA seq transcriptome sequencing:

[0045] After enriching eukaryotic mRNA with poly A tails by magnetic beads with Oligo (dT), the mRNA was cut by ultrasound. The fragmented mRNA was used as a template and random oligonucleotides as primers to synthesize the first cDNA chain in the M-MuLV reverse transcriptase system. Then, RNaseH was used to degrade the RNA chain, and the second cDNA chain was synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA was repaired at the end, A-tailed and connected to the sequencing adapter. AMPure XP beads were used to screen cDNA of about 200bp, PCR amplification was performed, and the PCR product was purified again using AMPure XP beads to finally obtain the library. (Technical support provided by Guangzhou Kidio Biotechnology Co., Ltd.)

[0046]

[0047] Table 2 Alpha diversity index of fecal microbiota of MIC

[0048] Through 16S rDNA high-throughput sequencing, the actual number of OTUs measured in the Control group, DSS group, and DSS+ARS group were 723 vs. 468 vs. 621, of which the DSS group and the Control group had 21 species in common, the DSS+ARS group and the Control group had 137 species in common, and the DSS+ARS group and the DSS group had 67 species in common. In α diversity, the Sobs, Chao 1, ACE, Shannon, and Simpson indices of each group are shown in Table 2, and the Shannon index is used as a representative bar graph. Due to factors such as large intra-group differences and small sample sizes, some indexes had no statistical differences, but the species diversity, richness, and uniformity of the intestinal flora of enteritis mice showed an improved trend, such as the attached Figure 3 As shown in A and B. Further, at the OTU level, the weighted_unifrac level of species evolutionary distance and species abundance was used to intuitively judge the β diversity of the intestinal flora of each group of mice. By drawing the UPGMA cluster tree and PCoA (PCo1+PCo2=70.04%) analysis, it can be seen that there are obvious differences in species structure among the three groups of mice. Among them, the intestinal flora characteristics of the Control group and the DSS group are relatively independent and the two-dimensional space distance is the farthest. The flora structure of the enteritis mice in the DSS+ARS group has a clear trend of moving closer to the Control group and away from the DSS group, as shown in the attached Figure 3 As shown in C and D.

[0049] At the phylum and genus levels, the top 10 species in relative abundance were selected to draw stacked graphs to show the enrichment of dominant species in each group, as shown in the attached figure. Figure 3E and F show the changes in the species composition of the intestinal flora of mice in each group as follows: At the phylum level, the intervention of artesunate mainly upregulated the DSS-induced

[0050] The average relative abundance of Firmicutes (52.49% vs. 23.92% vs. 42.90%; P = 0.0108), Patescibacteria (14.75% vs. 0.88% vs. 10.91%; P = 0.0068) and Tenericutes (0.60% vs. 0.01% vs. 0.18%; P = 0.0336) decreased, and the average relative abundance of Proteobacteria (11.69% vs. 43.05% vs. 12.66%; P = 0.0124) increased due to the downregulation of DSS.

[0051] Among them, as a sign of imbalance in intestinal flora homeostasis in IBD, the F / B (Firmicutes / Bacteroidetes) ratio was 3.22 vs. 1.39 vs. 0.82 in each group of mice, respectively. At the genus level, artesunate intervention mainly upregulated the decrease in the relative abundance of relatively beneficial bacteria such as Ruminococcaceae UCG-014 (7.19% vs. 0.20% vs. 8.85%; P = 0.0092) and Alistipes (8.42% vs. 0.46% vs. 1.44%; P = 0.0025) caused by DSS, and downregulated the decrease in the relative abundance of Bacteroides (1.21% vs. 23.76% vs. 13.60%; P = 0.0113) and Escherichia-Shigella (0.91% vs. 31.50% vs. 2.47%; P = 0.0113) caused by DSS. The relative abundance of conditional pathogens such as Erysipelatoclostridium (0.65% vs. 6.29% vs. 2.46%; P=0.0040) increased.

[0052] Embodiment 3

[0053] Microsoft Office Excel, IBM SPSS Statistics 26, Graphpad Prism8.0, and Adobe Illustrator 2023 were used for statistical analysis and graphics, and Image J was used to analyze relative grayscale values. Normally distributed quantitative data were expressed as mean ± standard deviation (SD); non-normally distributed quantitative data were expressed as median (upper quartile, lower quartile). The relative abundance of intestinal flora was expressed as percentage (%). Tukey's HSD test or Kruskal-Wallis H test was used to compare data between multiple groups, and Welch's t test or Wilcoxon rank sum test was used to compare data between two groups. P < 0.05 indicated that the difference was statistically significant, where ns indicated P > 0.05; * indicated P < 0.05; ** indicated P < 0.01; *** indicated P < 0.001; **** indicated P < 0.0001.

[0054] Using INDICATOR and LEfSe analysis, the attached Figure 4 As shown in the figure, the intestinal flora enriched species of enteritis mice after artesunate intervention included Candidatus Saccharimonas, Ruminococcaceae UCG-014, Alistipes, Enterorhabdus, Ruminiclostridium6, Ruminiclostridium 5 and Odoribacter at the genus level and Alistipes finegoldii at the species level. They are not only the indicator species with large and significant IndVal values ​​in the artesunate intervention group, but also the biomakers screened out with LDA (Linear Discriminant Analysis) scores ≥ 2 in LEfSe analysis.

[0055] By the attached Figure 5As shown in A and B, a total of 12,658 genes were detected by RNA seq in mouse colon tissue, and 115 differential genes were found, of which 45 genes were upregulated and 70 genes were downregulated after artesunate intervention. Among them, the top 30 differential genes with the smallest FDR value of the difference test include: Vnn1, Tlr7, Stip1, St8sia4, Scd2, Rgs13, Pof1b, Plin1, Phgdh, Mtm1, Marchf7, LPO, Il7r, Iglc3, Hsph1, Hspa1b, Hspa1a, Hsp90ab1, Gpd1, Fasn, Eif2s2, Cytip, COL5A3, Clec7a, Ccdc122, Casp4, Bmp5, Apobec1, Apoa1, Acaca. The top three differentially enriched KEGG signaling pathways were: PPARsignaling pathway (ko03320), AMPK signaling pathway (ko04152), and Estrogen signaling pathway (ko04915).

[0056] On this basis, the top three pathway-related genes were screened for joint omics analysis with intestinal flora, and a correlation heat map was made to show the top 100 genes and bacterial genera ranked by absolute value of correlation coefficient. Figure 5 As shown in C, among the target bacterial genus, Alistipes has a high correlation with differentially expressed genes related to PPAR, AMPK and Estrogen signaling pathways, and among the target pathways, the most bacterial groups are associated with differentially expressed genes related to the AMPK signaling pathway (Scd2, Fasn, Acaca, irs1, Creb3l1).

[0057] Example 4: Artesunate / Alistipes finegoldii upregulates the expression of AMPK / ACC

[0058] By the attached Figure 6As shown in the results, under anaerobic conditions, Alistipesfinegoldii proliferated in GAM anaerobic bacteria universal medium was directly gavaged into mice with DSS-induced acute enteritis. It was found that compared with the inflammation model group, the mice in the Alistipesfinegoldii intervention group had significantly reduced body weight, loose stools, bloody stools and other colitis symptoms that were significantly alleviated. At the end of the experiment, DAI was significantly reduced (P<0.001, P=0.0313), the degree of colon shortening (P<0.001, P=0.0162), and tissue pathological damage were significantly improved (P<0.0001; P=0.0009), and the activation of the classic inflammatory pathway NFκB was significantly reduced (P=0.0139; P=0.0136), which confirmed to a certain extent that the intestinal inflammation of mice was alleviated after gavage with bacterial solution.

[0059] In summary, using the DSS-induced acute enteritis model in mice, through DAI evaluation, HE, IHC, IF, RT-qPCR, WB and other methods, 16S rDNA, reference RNA seq transcriptome sequencing and omics joint analysis, we finally concluded that artesunate can effectively improve experimental acute enteritis in mice, and its anti-inflammatory mechanism may be achieved through the regulation of the microbiota, affecting the target functional bacteria Alistipes, and then promoting the expression of the AMPK / ACC pathway.

[0060] A person skilled in the art may understand that all or part of the steps in the method for implementing the above-mentioned embodiment may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, which, when executed, includes one or a combination of the steps of the method embodiment.

[0061] In addition, each functional unit in each embodiment of the present invention may be integrated into a processing module, or each unit may exist physically separately, or two or more units may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of a software functional module. If the integrated module is implemented in the form of a software functional module and sold or used as an independent product, it may also be stored in a computer-readable storage medium.

[0062] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Application of artesunate in improving ulcerative colitis via the Alistipes-AMPK axis.

2. The use of artesunate in improving ulcerative colitis via the Alistipes-AMPK axis according to claim 1, characterized in that: The Alistipes species is the Alistipes finegoldii strain.

3. A pharmaceutical composition for treating ulcerative colitis, characterized in that: It includes artesunate and at least one Alistipes probiotic and an AMPK pathway activator.

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