Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (4): 676-685.DOI: 10.19852/j.cnki.jtcm.2023.04.001
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Efficacy of Heshouwu (Radix Polygoni Multiflori) on gut mircobiota in mice with autoimmune encephalomyelitis
ZHOU Jun1, WANG Junhua2, LI Xiaobing2, WAN Chenyi2, LI Fangjun2, Lü Yanni3, CHEN Hao2(
), SUN Meiying2()
- 1 Department of Chinese Medicine, Gaoxin Branch of the First Affiliated Hospital of Nanchang University, Nanchang 330000, China
2 Department of Neurology, Gaoxin branch of the First Affiliated Hospital of Nanchang University, Nanchang 330000, China
3 Department of Pharmacy, Gaoxin branch of the First Affiliated Hospital of Nanchang University, Nanchang 330000, China
-
Received:2022-10-22Accepted:2023-03-04Online:2023-08-15Published:2023-07-03 -
Contact:CHEN Hao, Department of Neurology, Gaoxin branch of the First Affiliated Hospital of Nanchang University, Nanchang 330000, China. 15179132576@163.com. Telephone: +86-15113826448
SUN Meiying, Department of Neurology, Gaoxin branch of the First Affiliated Hospital of Nanchang University, Nanchang 330000, China. smy19871227@163.com. -
Supported by:National Natural Science Foundation of China: Role and Mechanism of Akkermansia-Mediated Activation of Microglia NLRP3 Inflammasome in Multiple Sclerosis(82101419);Jiangxi Provincial Natural Science Foundation: the Role and Mechanism of p38 MAPK Signaling Pathway Induced by Intestinal Flora Change in EAE Mice(20212BAB216024);Jiangxi Science and Technology Project of Chinese Medicine: Study on the Role and Mechanism of CGAS-STING Signaling Pathway in Microglia Mediated by Rhodiola Sachalinensis in Multiple Sclerosis(2021B660);Jiangxi Science and Technology Project of Chinese Medicine: Study on the Role and Mechanism of CGAS-STING Signaling Pathway in Microglia Mediated by Rhodiola Sachalinensis in Multiple Sclerosis(2022B1007);Jiangxi Science and Technology Program of Health Commission: the Role and Mechanism of Intestinal Flora Changes in Mediating the Activation of NLRP3 Inflammasome in Microglia in Multiple Sclerosis(202210392);Science and Technology Research Project of Jiangxi Education: Study on the Role and Mechanism of p38 MAPK Signaling Pathway Induced by Intestinal Flora Changes in EAE Mice(GJJ200215);Scientific Research and Development Project of the First Affiliated Hospital of Nanchang University: Effects of Exercise Training on Intestinal Flora of Multiple Sclerosis Model Rats and Its Mechanism(YFYPY202021)
Cite this article
ZHOU Jun, WANG Junhua, LI Xiaobing, WAN Chenyi, LI Fangjun, Lü Yanni, CHEN Hao, SUN Meiying. Efficacy of Heshouwu (Radix Polygoni Multiflori) on gut mircobiota in mice with autoimmune encephalomyelitis[J]. Journal of Traditional Chinese Medicine, 2023, 43(4): 676-685.
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Figure 1 Heshouwu (Radix Polygoni Multiflori) ameliorated the severity of EAE mice A: schematic timeline for the experimental procedures, at baseline (T1) mice in P. multiflorum group were administered orally with Heshouwu (Radix Polygoni Multiflori, 100 mg·kg-1·d-1) and Ad libitum group were treated with vehicle buffer for 2 weeks. (T2) Thereafter, all mice were injected intraperitoneally with PTX (300 mg, resuspended in 100 μL phosphate-buffered saline) and MOG35-55 peptide in CFA for immunization 7 d additionally, (T3) clinical EAE. B: EAE clinical score was assessed in mice treated with either vehicle buffer (ad libitum) or different concentration of P. multiforum (50, 100 and 200 mg·kg-1·d-1) daily and shown (n = 7). Three independent experiments were performed. Data represent mean ± standard error of mean. a: ns: no significance, compared with the ad libitum group. bP < 0.01, compared with the ad libitum group. cP < 0.01, compared with the ad libitum group. C1: quantification of inflammation in the lumbosacral spinal cords in the groups of mice treated with either vehicle buffer (ad libitum) or P. multiforum (100 mg·kg-1·d-1) (n = 6) on day 21 post-immunization. C2: quantification of demyelination in the lumbosacral spinal cords in the groups of mice treated with either vehicle buffer (ad libitum) or P. multiforum (100 mg·kg-1·d-1) (n = 6) on day 21 post-immunization. Data represent mean ± standard deviation. eP < 0.05, compared with the ad libitum group. dP < 0.01, compared with the Ad libitum group. D1-E1: hematoxylin-eosin staining of lumbar spinal cord sections in groups of mice treated with either vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum (scale bar 200 μm, magnification: ×100). D2-E2: scale bar 100 μm, magnification: ×200. F1-G1: luxol fast blue staining of lumbar spinal cord sections in groups of mice treated with either vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum (scale bar 200 μm, magnification: ×100). F2-G2: scale bar 100 μm, magnification: ×200. EAE: experimental autoimmune encephalomyelitis; PTX: pertussis toxin; CFA: complete Freund’s adjuvant; P. multiforum: Heshouwu (Radix Polygoni Multiflori).
Figure 1 Heshouwu (Radix Polygoni Multiflori) ameliorated the severity of EAE mice A: schematic timeline for the experimental procedures, at baseline (T1) mice in P. multiflorum group were administered orally with Heshouwu (Radix Polygoni Multiflori, 100 mg·kg-1·d-1) and Ad libitum group were treated with vehicle buffer for 2 weeks. (T2) Thereafter, all mice were injected intraperitoneally with PTX (300 mg, resuspended in 100 μL phosphate-buffered saline) and MOG35-55 peptide in CFA for immunization 7 d additionally, (T3) clinical EAE. B: EAE clinical score was assessed in mice treated with either vehicle buffer (ad libitum) or different concentration of P. multiforum (50, 100 and 200 mg·kg-1·d-1) daily and shown (n = 7). Three independent experiments were performed. Data represent mean ± standard error of mean. a: ns: no significance, compared with the ad libitum group. bP < 0.01, compared with the ad libitum group. cP < 0.01, compared with the ad libitum group. C1: quantification of inflammation in the lumbosacral spinal cords in the groups of mice treated with either vehicle buffer (ad libitum) or P. multiforum (100 mg·kg-1·d-1) (n = 6) on day 21 post-immunization. C2: quantification of demyelination in the lumbosacral spinal cords in the groups of mice treated with either vehicle buffer (ad libitum) or P. multiforum (100 mg·kg-1·d-1) (n = 6) on day 21 post-immunization. Data represent mean ± standard deviation. eP < 0.05, compared with the ad libitum group. dP < 0.01, compared with the Ad libitum group. D1-E1: hematoxylin-eosin staining of lumbar spinal cord sections in groups of mice treated with either vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum (scale bar 200 μm, magnification: ×100). D2-E2: scale bar 100 μm, magnification: ×200. F1-G1: luxol fast blue staining of lumbar spinal cord sections in groups of mice treated with either vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum (scale bar 200 μm, magnification: ×100). F2-G2: scale bar 100 μm, magnification: ×200. EAE: experimental autoimmune encephalomyelitis; PTX: pertussis toxin; CFA: complete Freund’s adjuvant; P. multiforum: Heshouwu (Radix Polygoni Multiflori).
Table 1 Changes in serum levels of cytokines at T1 and T3
| Item | T1 | T3 | |||
|---|---|---|---|---|---|
| ad libitum (n = 7) | P. multiforum (n = 7) | ad libitum (n = 7) | P. multiforum (n = 7) | ||
| GM-CSF | 1.70±0.17 | 1.72±0.23 | 3.31±0.23 | 2.20±0.27a | |
| IL-10 | 3.39±0.23 | 3.65±0.21 | 2.14±0.14 | 2.85±0.25b | |
| IL-21 | 7.11±0.32 | 7.24±0.29 | 4.51±0.36 | 6.28±0.29a | |
| IL-17A | 4.39±0.20 | 4.79±0.46 | 12.71±0.78 | 8.85±0.74a | |
| IL-17F | 3.41±0.21 | 3.29±0.23 | 7.88±0.36 | 5.77±0.32a | |
| IL-22 | 48.40±2.19 | 50.95±1.64 | 125.37±3.90 | 102.00±4.83a | |
Table 1 Changes in serum levels of cytokines at T1 and T3
| Item | T1 | T3 | |||
|---|---|---|---|---|---|
| ad libitum (n = 7) | P. multiforum (n = 7) | ad libitum (n = 7) | P. multiforum (n = 7) | ||
| GM-CSF | 1.70±0.17 | 1.72±0.23 | 3.31±0.23 | 2.20±0.27a | |
| IL-10 | 3.39±0.23 | 3.65±0.21 | 2.14±0.14 | 2.85±0.25b | |
| IL-21 | 7.11±0.32 | 7.24±0.29 | 4.51±0.36 | 6.28±0.29a | |
| IL-17A | 4.39±0.20 | 4.79±0.46 | 12.71±0.78 | 8.85±0.74a | |
| IL-17F | 3.41±0.21 | 3.29±0.23 | 7.88±0.36 | 5.77±0.32a | |
| IL-22 | 48.40±2.19 | 50.95±1.64 | 125.37±3.90 | 102.00±4.83a | |
Figure 2 Heshouwu (Radix Polygoni Multiflori) alleviated Th17/Treg imbalance in inguinal LN of EAE mice A: representative flow cytometric analysis (A1) and the percentage (right) of CD4+ T cells producing IL-17A (A2), GM-CSF (A3), IFN-γ (A4) in the CNS 21 d after immunization was shown (n = 6). aP < 0.01, frequency of IL-17A+ CD4+ T cells were compared with the ad libitum group. bP < 0.05, frequency of GM-CSF+ CD4+ T cells were compared with the ad libitum group. c: ns, frequency of IFN-γ+ CD4+ T cells were compared with the ad libitum group. B: representative flow cytometric analysis (B1) and the percentage (right) of CD4+ T cells producing IL-17A (B2), GM-CSF (B3), IFN-γ (B4) in the inguinal LN 21 d after immunization was shown (n = 6). dP < 0.01, frequency of IL-17A+ CD4+ T cells were compared with the ad libitum group. e: ns, frequency of GM-CSF+ CD4+ T cells were compared with the ad libitum group. f: ns, frequency of IFN-γ+ CD4+ T cells were compared with the ad libitum group. C: representative flow cytometric analysis (C1) and the percentage (C2) of CD4+ T cells producing Foxp3 in the CNS 21 d after immunization was shown (n = 6). g: ns, frequency of Foxp3+ CD4+ T cells were compared with the ad libitum group. D: representative flow cytometric analysis (D1) and the percentage (D2) of CD4+ T cells producing Foxp3 in the inguinal LN 21 d after immunization was shown (n = 6). hP < 0.01, frequency of Foxp3+ CD4+ T cells were compared with the ad libitum group. CNS: central nervous system; ns: no significance; LN: lymph node. IL-17: interleukin 17; IFN-γ: interferon-γ; GM-CSF: granulocyte-macrophage colony stimulating factor.
Figure 2 Heshouwu (Radix Polygoni Multiflori) alleviated Th17/Treg imbalance in inguinal LN of EAE mice A: representative flow cytometric analysis (A1) and the percentage (right) of CD4+ T cells producing IL-17A (A2), GM-CSF (A3), IFN-γ (A4) in the CNS 21 d after immunization was shown (n = 6). aP < 0.01, frequency of IL-17A+ CD4+ T cells were compared with the ad libitum group. bP < 0.05, frequency of GM-CSF+ CD4+ T cells were compared with the ad libitum group. c: ns, frequency of IFN-γ+ CD4+ T cells were compared with the ad libitum group. B: representative flow cytometric analysis (B1) and the percentage (right) of CD4+ T cells producing IL-17A (B2), GM-CSF (B3), IFN-γ (B4) in the inguinal LN 21 d after immunization was shown (n = 6). dP < 0.01, frequency of IL-17A+ CD4+ T cells were compared with the ad libitum group. e: ns, frequency of GM-CSF+ CD4+ T cells were compared with the ad libitum group. f: ns, frequency of IFN-γ+ CD4+ T cells were compared with the ad libitum group. C: representative flow cytometric analysis (C1) and the percentage (C2) of CD4+ T cells producing Foxp3 in the CNS 21 d after immunization was shown (n = 6). g: ns, frequency of Foxp3+ CD4+ T cells were compared with the ad libitum group. D: representative flow cytometric analysis (D1) and the percentage (D2) of CD4+ T cells producing Foxp3 in the inguinal LN 21 d after immunization was shown (n = 6). hP < 0.01, frequency of Foxp3+ CD4+ T cells were compared with the ad libitum group. CNS: central nervous system; ns: no significance; LN: lymph node. IL-17: interleukin 17; IFN-γ: interferon-γ; GM-CSF: granulocyte-macrophage colony stimulating factor.
Figure 3 Heshouwu (Radix Polygoni Multiflori) reconstituted the composition and diversity of gut microbiota in mice The clearance of intestinal flora by cocktail antibiotics was verified by 16s rRNA sequencing analysis. A: genus species phylogeny tree. B: PCoA plot shows the gut microbiota composition in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. C: relative abundances of the gastric microbiota at phylum (C1) and genus (C2) level in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. D: relative abundances of Bacteroidetes phylum (D1) and Firmicutes phylum (D2) in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. aP < 0.01, relative abundances of Bacteroidetes phylum were compared with the ad libitum group. bP < 0.01, relative abundances of Firmicutes phylum were compared with the ad libitum group. E: cladogram representation of the genus species in gastric microbiota in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. F: identify the specific microbiota taxa that significantly associated with 100 mg·kg-1·d-1 P. multiforum treatment by LEfSe. EAE: experimental autoimmune encephalomyelitis; P. multiforum: Heshouwu (Radix Polygoni Multiflori); PCoA: principal coordinate analysis; FMT: fecal microbiota transplantation; LDA: linear discriminant analysis; LEfSe: LDA effect size.
Figure 3 Heshouwu (Radix Polygoni Multiflori) reconstituted the composition and diversity of gut microbiota in mice The clearance of intestinal flora by cocktail antibiotics was verified by 16s rRNA sequencing analysis. A: genus species phylogeny tree. B: PCoA plot shows the gut microbiota composition in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. C: relative abundances of the gastric microbiota at phylum (C1) and genus (C2) level in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. D: relative abundances of Bacteroidetes phylum (D1) and Firmicutes phylum (D2) in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. aP < 0.01, relative abundances of Bacteroidetes phylum were compared with the ad libitum group. bP < 0.01, relative abundances of Firmicutes phylum were compared with the ad libitum group. E: cladogram representation of the genus species in gastric microbiota in group of mice treated with vehicle buffer (ad libitum) or 100 mg·kg-1·d-1 P. multiforum. F: identify the specific microbiota taxa that significantly associated with 100 mg·kg-1·d-1 P. multiforum treatment by LEfSe. EAE: experimental autoimmune encephalomyelitis; P. multiforum: Heshouwu (Radix Polygoni Multiflori); PCoA: principal coordinate analysis; FMT: fecal microbiota transplantation; LDA: linear discriminant analysis; LEfSe: LDA effect size.
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