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. 2023 Oct 17:11:e16226.
doi: 10.7717/peerj.16226. eCollection 2023.

Ginsenoside Rb1 enhanced immunity and altered the gut microflora in mice immunized by H1N1 influenza vaccine

Affiliations

Ginsenoside Rb1 enhanced immunity and altered the gut microflora in mice immunized by H1N1 influenza vaccine

Chuanqi Wan et al. PeerJ. .

Abstract

Background: Influenza is an acute infectious respiratory disease caused by the influenza virus that seriously damages human health, and the essential way to prevent influenza is the influenza vaccine. Vaccines without adjuvants produce insufficient specific antibodies and therefore require adjuvants to boost antibody titers. Microbes and hosts are a community that needs to "promote bacteria," which could provide new value for the immune effect.

Methods: (1) The H1N1 influenza vaccine, in combination with Ginsenoside Rb1, was co-injected into mice intraperitoneally (I.P.). Then, immunoglobulin G and antibody subtype levels were tested by enzyme-linked immunosorbent assay (ELISA). Moreover, mice were infected with a lethal dose of the H1N1 influenza virus (A/Michigan/45/2015), and survival status was recorded for 14 days. Lung tissues were stained by hematoxylin and eosin (H&E), and ELISA detected inflammatory factor expression levels. (2) Mice were immunized with Ginsenoside Rb1 combined with quadrivalent influenza inactivated vaccine(IIV4), and then IgG levels were measured by ELISA. (3) Fresh stool was collected for fecal 16S rDNA analysis.

Results: Ginsenoside Rb1 boosted IgG and antibody subtypes in the H1N1 influenza vaccine, improved survival of mice after virus challenge, attenuated lung histopathological damage, and reduced inflammatory cytokines expression in IL-6 and TNF-α. The results of 16S rDNA showed that Rb1 decreased species diversity but increased species richness compared to the PBS group and increased the abundance of Akkermansiaceae and Murbaculaceae at the Family and Genus levels compared with the HA+Alum group.

Conclusion: Ginsenoside Rb1 has a boosting effect on the immune efficacy of the H1N1 influenza vaccine and is promising as a novel adjuvant to regulate the microecological balance and achieve an anti-infective effect.

Keywords: 16S rRNA; Adjuvant; Ginsenoside Rb1; Gut microbiota; Polysaccharide.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Adjuvant effects of Ginsenoside Rb1 or Alum in combination with HA antigen.
PBS or 3 µg of antigens with or without adjuvant (Alum-100 µg, Rb1-70 µg, Rb1-200 µg, Rb1-600 µg) were injected into mice via I.P. route twice every two weeks. The vaccinated mice were challenged with 5×MLD50 of the virus (H1N1, A/Michigan/45/2015). (A) Vaccination, blood collection, feces collection, and challenge schedule. (B) IgG antibody response to H1N1 antigen. (C–E) IgG antibody subtype response to H1N1 antigen. The endpoint dilution of each sample was expressed as the absorbance value close to 2.1 times that of the negative control group. (F–G) The weight changes and survival rates against the virus. The dashed line means losing 30% weight loss. (* P < 0.05, ** P < 0.01).
Figure 2
Figure 2. Ginsenoside Rb1 attenuates severe lung injury and TNF-α and IL-6 levels during influenza virus infection.
Images are lung histopathology at day 6 of infection, 10× magnification (scale bar = 200 µm). (A) PBS (the model group). (B) H1N1 influenza vaccine-3ug(HA group). (C) H1N1 influenza vaccine+Alum adjuvant(HA+Alum group). (D) H1N1 influenza vaccine+Rb1-70 µg (HA+Rb1-70 ug). (E) H1N1 influenza vaccine+Rb1-200 µg (HA+Rb1-200ug). (F) H1N1 influenza vaccine+Rb1-600 µg (HA+Rb1-200ug). (G, H) Cytokine expression of TNF-α and IL-6 were measured in serum from mice six days after virus infection, n = 3–4. (*P < 0.05).
Figure 3
Figure 3. Adjuvant effect of Ginsenoside Rb1 on IIV4.
(A) Vaccination, blood collection. PBS or 6ug of IIV4 (1.5 ug of each antigen derived from four strains) with or without Rb1 were once injected into mice via the I.P. route. Sera were collected three weeks after the vaccination. (B–E) Serum IgG titers for four different influenza strains (n = 6).
Figure 4
Figure 4. Ginsenoside Rb1 altered fecal microbial diversity in immunized mice.
16S rRNA sequencing of the fecal samples in control (PBS), the model (HA), the Alum administration (HA+Alum), and the Rb1 administration groups (HA+Rb1) were detected at 23 dpi (n = 6). Alpha-Diversity was evaluated based on the Shannon (A), Simpson (B), Chao1 (C), and ACE (D) indices of the operational taxonomic unit (OTU) levels. Beta-Diversity was evaluated based on the principal coordinate analysis (PCoA) (E, F).
Figure 5
Figure 5. Ginsenoside Rb1 altered the dominant bacteria and microbiota composition in immunized mice.
The community abundance at the Family (A) and Genus (B) levels in control (PBS), the model (HA), the Alum administration (HA+Alum), and the Rb1 administration groups (HA+Rb1). (C) Species marker evolution trees for each group. The circles represent, from inside to outside, the taxonomic levels by phylum to genus (or species). Circle diameter size is proportional to relative abundance size. Species with no significant differences are colored in yellow, and the group colors those with differences.
Figure 6
Figure 6. Biomarkers with significant abundance differences among groups (LAD value > 2).
Column lengths represent the contribution of biomarker species.

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Grants and funding

This work was supported by the Science and Technology Project of Zhejiang Province (Grant no. 2023ZL047), and the Key Research Projects of Zhejiang Chinese Medical University (Grant no. 2022FSYYZZ03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.