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Authors: Wang Tao, Dai Mingzhu, Li Yanchuan, Peng Yi, Li Chunqi author: Infinitus (China) Co.
, Ltd.
; Hangzhou Huante Biotechnology Co.
, Ltd.
This article was published in: Chinese Journal of Laboratory Animals, January 30, 2022 Core Abstract: Objective: To rapidly evaluate the effect of compound Chinese medicine extract on enhancing immunity through in vitro cell and in vivo zebrafish experiments, and to conduct preliminary mechanism research
。 Methods: The biological activity of compound Chinese medicine extract was preliminarily evaluated by RAW 264.
7 mouse monocytic macrophage leukemia cells in vitro experiments.
Then, vinorelbine tartrate was used to induce the establishment of a model of in vivo immunodeficiency in zebrafish, and the intervention effect
of compound Chinese medicine extracts was observed from the aspects of macrophage production and phagocytosis of zebrafish, T cell production and related inflammation, and expression of immune genes.
Results: In vitro experiments, the compound Chinese herbal extract had good biological safety, and it could improve the survival rate
of RAW 264.
7 mouse mononuclear macrophages induced by vinorelbine tartrate at 250 μg/mL 。 In vivo studies have shown that compound Chinese herbal extracts can significantly improve the reduction of the number of zebrafish macrophages induced by vinorelbine at concentrations of 222, 667 and 2000 μg/mL, and enhance macrophage phagocytosis.
The concentration of 222 and 667μg/mL can significantly increase the fluorescence intensity of T cells.
The concentrations of 222, 667 and 2000 μg/mL can significantly restore the relative expression of rag1 and rag2 genes in the model control group, and can significantly reduce the relative expression of TNF-α gene in model zebrafish.
Compound Chinese herbal extract 222μg/mL can significantly upregulate the expression of immune factor IL-12 gene, and significantly upregulate IFN-γ gene expression at 2000μg/mL
.
Conclusion: In vivo and in vitro experiments show that compound Chinese medicine extracts have obvious immune-enhancing effects
.
● Review immunity can protect the body from infringement, in recent years, due to ecological imbalance caused by more and more immune diseases, Western medicine can not play a preventive role, the treatment effect is not very obvious and there are toxic side effects
.
Under the guidance of the basic theory of traditional Chinese medicine, Chinese medicine and its extracts are good at treating diseases, pay attention to coordination and balance, and have few toxic and side effects, so Chinese medicine and its extracts have always been a new research direction
to enhance immunity.
Experimental studies have shown that Ganoderma lucidum polysaccharides and alkaloids can significantly enhance the phagocytosis ability and T lymphocyte transformation ability of mouse macrophages, and can increase the expression level
of IFN-γ in the peripheral blood of mice.
Schisandra polysaccharides, Baishu polysaccharides and lactones all have antioxidant and enhance immunity activities; Dodder extract is a potent antibacterial agent, antioxidant and anti-inflammatory; Codonopsis ginseng and poria have shown anti-inflammatory activity
in different experimental models of multiple acute and chronic inflammations.
However, most of the studies are evaluated on traditional mice as animal models, which has the disadvantages
of long molding time, high cost and low throughput.
Zebrafish is an internationally recognized novel model organism whose compelling advantages are the transparency of embryonic development, a high-throughput detection system, and short-efficient-low-cost screening
.
With the implementation and implementation of the "3R principle", it has been widely
used in drug target identification, disease model development and toxicity evaluation.
In this study, the biosafety and immune enhancement effects of compound Chinese medicine extracts were investigated from the aspects of RAW264.
7 mouse mononuclear macrophage leukemia cell activity, the number of zebrafish macrophages and their phagocytic function, the number of T cells, and the level of immune-related genes, and the compound Chinese medicine extracts to verify and provide theoretical basis
。 ● Experimental method 1.
RAW264.
7 cell culture and compound Chinese medicine extract treatment took RAW264.
7 mouse mononuclear macrophage leukemia cells with good growth state, and added medium with concentrations of 62.
5, 125, 250, 500 and 1000μg/mL of compound Chinese medicine extract to each well, and determined the absorbance (OD) value of each pore to determine the treatment concentration of compound Chinese medicine extract.
Except for the normal control well and the solvent control well (0.
1% DMSO), the medium with a concentration of 12.
5μg/mL was added to each well, and then the medium of 27.
8, 83.
3, 250μg/mL of compound Chinese medicine extract and 25μg/mL concentration of positive control group were added in order to determine the OD value of each well, and the effect of compound Chinese medicine extract on the survival rate of
RAW264.
7 cells was evaluated 。 2.
The safety investigation of compound Chinese medicine extract selected 2dpf Tg (mpeg1:EGFP) transgenic zebrafish after fertilization, and set up dosing group: compound Chinese medicine extract 62.
5, 125, 250, 500, 1000, 2000μg/mL; Set up a normal control group
at the same time.
The toxic phenotype and mortality of zebrafish organs at incubation to 6 dpf were recorded, and the detectable concentration (MTC)
of compound Chinese herbal extracts for normal zebrafish was determined.
3.
The effect of compound Chinese medicine extract on the production of zebrafish macrophages was taken from 2dpf Tg transgenic zebrafish, and the dosing group was set: compound Chinese medicine extract 222, 667, 2000μg/mL; At the same time, a control group was set up to establish a model
of macrophage in zebrafish.
Observe macrophage fluorescence intensity (S1) in the venous vessels of the tail of zebrafish under a fluorescence microscope and take pictures
.
4.
The effect of compound Chinese medicine extract on the phagocytosis function of zebrafish macrophages was selected 2dpfTg transgenic zebrafish, and red fluorescent microspheres were injected intravenously as foreign substances to establish a model
of zebrafish macrophage phagocytosis.
Set the model zebrafish dosing group: compound Chinese medicine extract 222, 667, 2000 μg/mL; At the same time, a control group was set up to observe the number (N) of fluorescent microspheres in the zebrafish (whole-body)
under a fluorescence microscope.
5.
The effect and mechanism of compound Chinese medicine extract on zebrafish T cells and preliminary mechanism selected 2dpf Tg transgenic zebrafish and set up the dosing group: compound Chinese medicine extract 222, 667, 2000 μg/mL; At the same time, the control group was set: positive control Bailing capsule 60.
0 μg/mL, normal control group and model control group, and a zebrafish T cytopenia model
was established 。 Zebrafish T cell fluorescence intensity (S2) was observed under a fluorescence microscope and the effect on T cell production was analyzed; Zebrafish samples were collected, total zebrafish RNA of each concentration group was extracted, and β-actin was used as an internal reference for gene expression to detect tumor necrosis factor-α (TNF-α, TNF-α), recombinant-activated geno-1 (rag1), recombinant-activated geno-2 (rag2), interleukin-12 (IL-12) and γ-interferon-γ, IFN-γ), and the initial mechanism of compound Chinese medicine extracts to enhance epidemic immunity was
studied.
● Experimental results 1.
Effect of Compound Chinese Medicine Extract on RAW 264.
7 Cell Survival When the concentration of compound Chinese medicine extract was 62.
5~250μg/mL, the survival rate of RAW264.
7 cells > 90.
0%; When the concentration of compound herbal extract was 500μg/mL, the viability of RAW264.
7 cells decreased to 84.
0%.
Therefore, the safe concentration of compound Chinese herbal extract for RAW264.
7 cells was determined to be 250μg/mL (see Table 1).
It can be seen from Table 2 that vinpo rubine tartrate can lead to a significant decrease in the survival rate of RAW264.
7 cells, and the cell survival rate increases in a dose-dependent manner after the intervention of 3-fold concentration gradient of compound Chinese medicine extract, and 250μg/mL of compound Chinese medicine extract can significantly improve the survival rate of RAW264.
7 cells (P<0.
01).
2.
At the end of the experiment, there were no obvious abnormalities in zebrafish in the concentration group of compound Chinese medicine extract 62.
5~2000μg/mL, and the organs and behavioral status were similar to normal zebrafish (see Figure 1).
It was suggested that the MTC of compound Chinese medicine extract for normal zebrafish was 2000μg/mL
.
3.
The effect of compound Chinese medicine extract on the generation of macrophages in zebrafish can be seen from the statistical results (Table 3, Figure 2), vinporebine tartrate can lead to low immunity in zebrafish, which is manifested as a significant decrease in macrophage fluorescence intensity in zebrafish in vinpo tartrate group compared with normal control group; After the intervention of the three-fold concentration gradient of low, medium and high concentrations of compound Chinese medicine extracts, the fluorescence intensity of macrophages increased significantly, and the improvement effect of macrophage reduction was 18% (P< 0.
05), 21% (P<0.
01) and 22% (P<0.
01),
respectively 。 4.
The effect of compound Chinese medicine extract on the phagocytic function of zebrafish macrophages can be seen from the statistical results (Table 4, Figure 3), compared with the model control group, the three concentration groups of compound Chinese medicine extract can significantly reduce the number of fluorescent microspheres in zebrafish (P<0.
05), indicating that the compound Chinese medicine extract has the effect
of promoting macrophage phagocytosis 。 5.
The effect of compound Chinese herbal extract on zebrafish T cell production can be seen from the statistical results (Table 5, Figure 4), compared with the normal control group, the fluorescence intensity of T cells in the model control group is significantly reduced, indicating that vinporebine tartrate can induce zebrafish immunosuppression
.
Compared with the model control group, the concentration of T cells in the compound Chinese medicine extract was fluorescenceThe intensity increased significantly (P<0.
01); However, the high concentration did not increase the fluorescence intensity of T cells and showed a decreasing effect
.
6.
Effect and preliminary mechanism of compound Chinese medicine extract on zebrafish T cells 6.
1 RNA concentration and purity were extracted at the end of the experiment, and the total RNA of zebrafish was extracted to determine the RNA concentration and purity, and the results showed that the A260/A280 ratio was between 1.
88~2.
03, indicating that the total RNA extracted from zebrafish was of good quality and could be used for subsequent q-PCR experiments (see Table 6).
。 6.
2 The effects on the recombinant-activated gene rag1 and rag2 were calculated according to the formula of relative expression of genes, and compared with the normal control group, the relative expression of rag1 and rag2 genes in the model control group was significantly reduced (P<0.
01), and the relative expression of rag1 and rag2 genes in the low, medium and high concentration groups was significantly restored after the intervention of compound Chinese medicine extract (see Table 7).
。 6.
3 Compared γ with the normal control group, the relative expression of IL-12 gene in the model control group was significantly lowered (P<0.
001), and after the intervention of compound Chinese medicine extract, the relative expression of IL-12 gene in the low-concentration group increased significantly (P < 0.
001), and negative feedback regulation
occurred in the medium- and high-concentration groups 。 Compared with the normal control group, the relative expression of IFN-γ gene in the model control group was significantly lowered (P<0.
01), and the relative expression of IFN-γ gene was dose-dependent after the intervention of 3-fold concentration gradient of compound Chinese medicine extract, and the relative expression of IFN-γ gene in the high concentration group of compound Chinese medicine extract increased significantly (P <0.
001) (see Table 8).
。 6.
4 The effect on the inflammatory factor TNF-α model The relative expression of TNF-α gene in the control group (1.
000) was significantly upregulated compared with that in the normal control group (0.
006) (P<0.
001).
The relative expression of TNF-α gene in the low, medium and high concentration groups of compound Chinese medicine extract was 0.
023, 0.
002 and 0.
008 (P<0.
001), respectively, indicating that the relative expression of TNF-α gene could be significantly reduced by the compound Chinese medicine extract (see Table 9).
● Conclusion In vitro experiments, compound Chinese medicine extracts have good biological safety, and can significantly improve the survival rate
of mouse mononuclear macrophages after induction of vinorelbine tartrate 。 In vivo studies have shown that compound Chinese herbal extracts can significantly improve the decrease in the number of zebrafish macrophages and T cells induced by vinorelbine, and enhance the activity and phagocytosis of macrophages in vivo.
Compound Chinese medicine extract can significantly restore the relative expression of rag1 and rag2 genes in the model control group, compound Chinese medicine extract can upregulate the expression of immune factor IL-12 and IFN-γ gene, and compound Chinese medicine extract can significantly reduce the relative expression of
TNF-α gene.
In vivo and in vitro experiments have shown that compound Chinese medicine extracts have obvious immune-enhancing effects
.
In summary, compound Chinese herbal extracts have obvious anti-inflammatory and immune-enhancing effects
.
This study can provide a guiding basis
for the use of compound Chinese herbal extracts as new immune health foods and secondary development.