Effects of Scutellaria baicalensis Extract on Gut Microbiota Diversity in a Heat-Induced Rat Model Based on High-Throughput Sequencing
Release Date:
2021-02-02
Impact of Scutellaria baicalensis Extract on Gut Microbiota Diversity in a Heat-Induced Rat Model Based on High-Throughput Sequencing
Abstract: Objective: To investigate Scutellaria. Scutellaria baicalensis To investigate the effects of the extract on intestinal microbiota diversity in a heat syndrome rat model and to explore the scientific implications of the TCM principle “treat heat with cold,” the following methods were employed: Male SD rats were randomly assigned to a control group, a model group, and a Scutellaria baicalensis extract group (3 g/kg). Except for the control group, all other groups received daily morning intragastric administration of levothyroxine sodium suspension (0.12 g/kg); 1 hour after modeling, the Scutellaria baicalensis extract group was administered the extract (10 mL/kg), while the control and model groups received an equal volume of distilled water, for a total of 15 consecutive days. High-throughput sequencing was used to analyze the diversity of the intestinal microbiota in the cecal contents of the rats; serum samples were collected to measure key indices of the neuro–endocrine–immune system, and a network of interactions between differentially abundant bacterial genera and the neuro–endocrine–immune system was constructed. The results showed that Scutellaria baicalensis extract significantly normalized 12 key indices of the neuro–endocrine–immune system in the heat syndrome model rats, markedly improved the dysregulation of operational taxonomic units (OTUs) and α- and β-diversity in the gut, reduced microbial abundance and diversity indices, and modulated the relative abundances of six differentially abundant phyla and 25 differentially abundant genera in the heat syndrome model rats. Specifically, the genus Collinsella Collinsella 1. Genus Paleobacter Ochrobactrum 1. Prevotella Prevotella It exhibits strong correlations with the neuro–endocrine–immune system. Conclusion: Baicalin extract can ameliorate the abnormalities in the neuro–endocrine–immune system and gut microbiota diversity in heat‑syndrome model rats, and the genera Collinsella, Alistipes, and Prevotella may serve as potential biomarker genera in the gut of these rats.
Heat syndrome is an important pattern in traditional Chinese medicine, with its etiology primarily attributed to excessive yang qi, pathogenic heat invading the blood, and deficiency of yin qi. It manifests as symptoms of hyperactive bodily energy, such as dry mouth, fever, and tachycardia. [1-2] In the treatment of heat syndromes with traditional Chinese medicine, the guiding principle is “cooling what is hot.” Research has shown that the neuro–endocrine–immune system serves as a critical target for regulating the onset and progression of these conditions. [3] Medicinal substances, via various pathways and characterized primarily by their effects on the neuroendocrine-immune system, can reveal the correlation between the targets of traditional Chinese medicines and the targets of TCM syndromes, thereby serving as an important manifestation of the TCM principle of “syndrome differentiation and individualized treatment.” The gut microbiota is closely associated with TCM syndromes. [4] . Zhang Beihua et al. [5] Research has shown that dysbiosis of the gut microbiota can lead to spleen deficiency–related diarrhea, thereby exacerbating the pathological manifestation of dampness obstruction in spleen deficiency syndrome; Huang Tengjie [6] It has been found that dysbiosis of the gut microbiota is closely associated with yang deficiency syndrome, and specific bacterial genera such as Roseburia have been identified as characteristic taxa of this condition. However, to date, no studies have reported whether there are differences in the functional diversity and structural composition of the host gut microbiota under febrile conditions compared with those in healthy individuals.
Scutellaria Scutellaria baicalensis Scutellaria baicalensis is cold in nature and bitter in taste, and it primarily enters the Lung, Gallbladder, Stomach, and Large Intestine meridians. In previous research, our team designated Scutellaria baicalensis as a cold-pattern herbal medicine and demonstrated that it exerts significant inhibitory effects on substance and energy metabolism as well as on related biochemical enzymes in heat-pattern rat models. [7] However, it remains unclear whether such interventions can exert effects on the neuro–endocrine–immune system. In this study, we employed Illumina MiSeq–based 16S rRNA high-throughput sequencing to investigate the changes in intestinal microbiota diversity and the associated impacts on the neuro–endocrine–immune system in a heat‑syndrome rat model following treatment with Scutellaria baicalensis extract. Pearson correlation analyses were conducted between differentially abundant bacterial genera and relevant neuro–endocrine–immune markers, with the aim of elucidating the mechanistic basis of Scutellaria’s intervention in heat syndrome from the perspective of the gut microbiota–host interaction. This research seeks to establish the interplay among host, microbiota, and syndrome patterns, thereby laying a theoretical foundation for refining the TCM syndrome evaluation system and further clarifying the scientific rationale underlying the principle “cooling the hot.”
1 Materials
1.1 Animal
Thirty SPF-grade male SD rats, 6–8 weeks old and weighing (180 ± 20) g, were provided by the Laboratory Animal Center of Heilongjiang University of Traditional Chinese Medicine, with license number SCXK (Hei) 2015-004. The animals were housed at the GLP Experimental Center of Heilongjiang University of Traditional Chinese Medicine under conditions of room temperature 20–25°C and relative humidity 40%–60%. They were fed standard laboratory chow and had free access to water, and the study was approved by the Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (approval number DXLL2015061601).
1.2 Pharmaceuticals and Reagents
Scutellaria baicalensis (batch number 20140623) was purchased from Hebei Chengde Medicinal Materials Co., Ltd. and identified by Professor Wang Zhenyue of the Teaching and Research Section of Chinese Medicinal Resources at Heilongjiang University of Traditional Chinese Medicine as Scutellaria baicalensis, a plant belonging to the Lamiaceae family. S. baicalensis Georgi’s dried root; levothyroxine sodium tablets (batch number H20160056, specification 50 μg/tablet) were purchased from Merck-Leon Pharmaceutical Company in Germany; rat norepinephrine (NE) ELISA kit, rat 5-hydroxytryptamine (5-HT) ELISA kit, rat dopamine (DA) ELISA kit, rat thyroid-stimulating hormone (TSH) ELISA kit, rat thyrotropin-releasing hormone (TRH) ELISA kit, rat adrenocorticotropic hormone (ACTH) ELISA kit, rat corticotropin-releasing hormone (CRH) ELISA kit, rat 17-hydroxycorticosteroids (17-OHCS) ELISA kit, rat interleukin-2 (IL-2) ELISA kit, rat interleukin-6 (IL-6) ELISA kit, rat interleukin-10 (IL-10) ELISA kit, and rat interferon-γ (IFN-γ) ELISA kit, all with batch number 20160816, were purchased from the Nanjing Jiancheng Institute of Biological Engineering; rat feed was purchased from Beijing Ke’ao Xielì Feed Co., Ltd.
1.3 Instrument
FLx800 multifunction microplate reader (BioTek, USA); RS232G UV–Vis spectrophotometer (Eppendorf, Germany); AUT0520 gel imaging system and 32RL117950 integrated UV analyzer (BG, USA); Pico17 centrifuge (Thermo, USA); QuantiFluor TBS380 fluorometer (Promega, USA).
2 Method
2.1 Preparation of Scutellaria baicalensis Extract
The processed Scutellaria baicalensis slices were soaked for 30 minutes, then extracted with distilled water at a solvent-to-material ratio of 10:1 and 8:1, respectively. The two extracts were combined, subjected to reflux condensation for 2 hours, and the extractive solution was recovered under reduced pressure. The resulting solution was then freeze-dried under vacuum to yield a dry powder (determined by HPLC to contain 12.87% baicalin, 0.51% baicalein, 3.36% wogonoside, and 0.28% wogonin), which was stored in a desiccator for later use.
2.2 Animal Grouping 、 Modeling and Drug Administration
After a 3-day acclimatization period, the rats were randomly assigned to a control group, a model group, and a baicalin extract group (3 g/kg), with 10 rats in each group, based on the principle of body-weight–based randomization. [8] Levothyroxine sodium tablets were ground into a powder and dissolved in drinking water to prepare a solution with a mass concentration of 0.04 g/mL; baicalin extract lyophilized powder was dissolved in drinking water to prepare a suspension with a mass concentration of 1 g/mL. At 9:00 each day, the control group was administered drinking water by gavage (10 mL/kg), while the other groups of rats were administered levothyroxine sodium suspension by gavage (0.12 g/kg). [9] From the onset of model induction, the baicalin extract group was administered baicalin extract by gavage at 10:00 daily (10 mL/kg), while the control and model groups were given an equal volume of distilled water by gavage, for a total of 15 consecutive days.
2.3 Vital Signs Monitoring
Daily, before drug administration, record changes in water intake and body weight; on days 0, 5, 10, and 15 of the dosing period, measure and record rectal temperature.
2.4 Sample Collection and Processing
Twenty-four hours after the last dose, rats were anesthetized with 20% urethane via intraperitoneal injection. Blood was collected from the abdominal aorta, allowed to stand at room temperature for 30 minutes, and then centrifuged at 4°C and 3,500 rpm for 10 minutes. The supernatant was collected and stored at −80°C for later use.
Five rats were randomly selected from each group; after cervical dislocation, the cecal contents were collected into sterile centrifuge tubes, rapidly frozen in liquid nitrogen, and immediately transferred to a −80°C freezer for subsequent analysis of gut microbial diversity.
2.5 Intestinal Microbiota Diversity Analysis
The Illumina MiSeq sequencing platform was used, with universal primers targeting the V4 variable region of the 16S rRNA gene for amplification. The primer sequences are 280 bp in length: the forward primer is 5’-AYTGGGYDTAAAGNG-3’, and the reverse primer is 5’-TACNVGGGTATCTAATCC-3’. The PCR program consisted of a 98°C pre-denaturation step for 30 seconds, followed by denaturation at 98°C for 15 seconds, annealing at 50°C for 30 seconds, and 25 cycles of these steps; extension was performed at 72°C for 30 seconds, with a final extension at 72°C for 5 minutes, followed by storage at 4°C. Amplified products were recovered using a gel extraction kit. Library quality assessment and quantification were then carried out on the PCR products using the Illumina MiSeq library quantification and quality control kit, after which libraries were prepared using a high-sensitivity DNA quantification kit. Sequencing was performed on the MiSeq platform using paired-end reads with a read length of 2 × 300 bp per cycle. Sequencing data were subjected to visual analysis using QIIME, R, and Mothur software.
2.6 Serum biomarker testing
The levels of NE, 5-HT, DA, TSH, TRH, ACTH, CRH, 17-OHCS, IL-2, IL-6, IL-10, and IFN-γ in rat serum were determined according to the instructions provided with the enzyme-linked immunosorbent assay kit.
2.7 Data Processing
All experimental data for each group were subjected to statistical analysis using SPSS 20.0. Data conforming to a normal distribution were expressed as mean ± standard deviation, and one-way ANOVA was performed. For pairwise comparisons among groups with homogeneity of variance, the least significant difference test was used; for those with heterogeneity of variance, Dunnett’s test was employed.
3 Result
3.1 Effects of Scutellaria baicalensis Extract on Physiological Parameters in a Heat-Excess Rat Model
As shown in Figure 1, on day 15, compared with the control group, the model group exhibited elevated rectal temperature, as well as significant increases in water intake and body weight ( P <0.01); compared with the model group, the rats in the Scutellaria baicalensis extract group exhibited decreased rectal temperature and significantly reduced water intake and body weight ( P (<0.01), indicating that the Scutellaria baicalensis extract can significantly ameliorate the alterations in general clinical signs in the heat syndrome model rats.
3.2 Effects of Scutellaria baicalensis Extract on Serum Parameters in a Heat-Induced Rat Model
As shown in Tables 1–3, compared with the control group, the model group exhibited significantly elevated serum levels of NE, DA, TSH, TRH, CRH, ACTH, 17-OHCS, IL-2, and IFN-γ ( P <0.05, 0.01), and the levels of 5-HT, IL-6, and IL-10 were significantly reduced ( P <0.01); compared with the model group, the baicalin extract group exhibited significantly reduced levels of NE, DA, TSH, TRH, CRH, ACTH, 17-OHCS, IL-2, and IFN-γ in rats ( P <0.05, 0.01), and the levels of 5-HT, IL-6, and IL-10 were significantly increased ( P <0.01).
3.3 Intestinal microbiota operational taxonomic unit ( operational taxonomic units , OTUs ) and diversity analysis
As shown in Figure 2, compared with the control group, the model group exhibited a significant increase in the relative abundance of OTUs at the phylum, class, order, family, genus, and species levels of the gut microbiota; in contrast, compared with the model group, the baicalin extract group showed a certain degree of reduction in the relative abundance of these same six taxonomic levels. As illustrated in Figure 3, as the number of samples increases, the total number of microbial taxa tends to stabilize at a constant value, indicating that the sequencing depth is sufficient and that the Specaccum species accumulation curve adequately reflects the community’s abundance profile. The results of the α-diversity analysis are presented in Table 4: compared with the control group, the model group showed significantly higher richness indices (Chao1 and ACE) and diversity indices (Simpson and Shannon) for the gut microbiota. P <0.01); compared with the model group, the abundance index and diversity index of the intestinal microbiota in the Scutellaria baicalensis extract group were significantly reduced ( P <0.01). Principal component analysis (PCA) was used to examine the differences in β-diversity of the gut microbiota in rats. As shown in Figure 4, the gut microbiota of the model group and the control group were clearly separated, indicating a significant difference in microbial community structure between the two groups. In contrast, the gut microbiota of the baicalin extract–treated group tended to cluster with that of the control group, suggesting that the microbial community structure in this group was similar to that of the control group, implying a possible phylogenetic relationship between the two.
3.4 Analysis of Gut Microbiota Composition and Structure
As shown in Figure 5, at the phylum level, compared with the control group, the model group of rats exhibited significant differences in six bacterial phyla in their gut microbiota ( P (<0.05, 0.01), the phyla Bacteroidetes, Spirochaetes, Proteobacteria, and Actinobacteria were upregulated, while the phyla Firmicutes and Fusobacteria were downregulated; the baicalin extract group significantly reversed the changes in these six phyla ( P <0.05, 0.01). As shown in Figures 6 and 7, at the genus level, cluster analysis was performed on the top 50 dominant genera among the differentially abundant phyla, and the positions of genera with common differential characteristics were annotated on the phylogenetic tree, namely the genus *Pseudomonas*. Atopobium 1. Coccus bacillus genus Aggregatibacter 1. Genus Blautia Blautia 1. Bacteroides Bacteroides , genus Christensenella Christensenella , Clostridium Clostridium 1. Collinsella Collinsella 1. Corynebacterium Corynebacterium 1. Genus Bacillus Coprobacillus 1. Genus Coprophilus Coprococcus 1. Dolosicoccus Dorea 1. Fusobacterium Fusobacterium 1. Genus Helicobacter Helicobacter 1. Genus Aerococcus Aerococcus 1. Genus Spirochaeta Oscillospira 1. Genus Paleobacter Ochrobactrum 1. Parabacteroides Parabacteroides 1. Genus *Koala* Phascolarctobacterium 1. Paraprevotella Paraprevotella 1. Prevotella Prevotella 1. Roseomonas Rothia 1. Genus Ruminococcus Ruminococcus 1. Sartorya Sutterella 1. Staphylococcus Staphylococcus 1. Treponema genus Treponema As shown in Figure 8, compared with the control group, the model group of rats exhibited significant differences in 25 bacterial genera in their gut microbiota ( P (<0.05, 0.01), with 17 bacterial genera upregulated and 8 downregulated. Compared with the model group, the Scutellaria baicalensis extract group significantly reversed the dysregulation of these 25 bacterial genera ( P <0.05, 0.01).
3.5 Association Analysis of Differential Gut Microbiota
Using Pearson correlation analysis, this study investigates the relationship between dysbiotic gut microbiota and the neuroendocrine-immune system during the intervention of Scutellaria baicalensis extract on serum biomarkers in a heat syndrome rat model, when | r |＞0.5 and P <0.05 indicates a significant correlation between the two; when | r |＞0.8 indicates a strong correlation between the two. As shown in Figures 9 and 10, the nervous system is strongly correlated with Prevotella, Collinsella, and Dorea; the immune system is strongly correlated with Prevotella, Collinsella, Helicobacter, Sutterella, Staphylococcus, Christensenella, and Acinetobacter; and the endocrine system is strongly correlated with Ruminococcus, Collinsella, Bacteroides, Clostridium, Aeromonas, Staphylococcus, and Acinetobacter. These findings suggest that Scutellaria baicalensis extract not only enhances gut microbiota diversity but also modulates key parameters of the neuro–endocrine–immune axis.
4 Discussion
In TCM clinical practice, “heat syndrome” is a complex and comprehensive umbrella term. According to TCM pattern differentiation, it is generally classified into excess heat syndrome and deficiency heat syndrome, with corresponding standardized evaluation and quantitative diagnostic criteria developed in conjunction with the affected body region or underlying etiology. [1] Levothyroxine sodium, also known as Euthyrox, is commonly used in clinical practice to treat hypothyroidism caused by various etiologies and can promote oxidative reactions in the body’s tissues and organs. [10] , changes in fluid intake, body weight, and body temperature are the direct manifestations of its general physical signs. [11] DA, 5-HT, and NE are important neurotransmitters in the central nervous system that regulate the functions of the sympathetic nervous system and the spinal nervous system, serving as key indicators of excitatory activity within the brain. [12] CRH, ACTH, and 17-OHCS are key adrenal cortical hormones involved in the hypothalamic–pituitary axis; together with TRH and TSH, they regulate the hypothalamus–pituitary–thyrotropin axis of the endocrine system, reflecting the hypermetabolic state associated with heat syndrome in Traditional Chinese Medicine. [13] In traditional Chinese medicine, heat syndromes accelerate the consumption of the immune system, increasing the conversion rate of T lymphocytes and leading to a decrease in CD4. plus IL-2, IFN-γ, and CD8 in T cells plus Dysregulation of IL-6 and IL-10 levels in T cells leads to elevated pro-inflammatory cytokine levels and reduced anti-inflammatory cytokine levels. [14-15] Based on the integrated analysis of dietary intake, rectal temperature, and body weight, the results of this study demonstrate that, under conditions of heat syndrome, all physiological systems of the organism are in a state of heightened activity. Moreover, the extract of Scutellaria baicalensis exerts regulatory effects on key indices within the neuroendocrine–immune system of heat-syndrome model rats, indicating that this extract can improve the neuroendocrine–immune system and associated clinical manifestations from a holistic perspective—thus embodying the traditional Chinese medicine principle of “cooling what is hot.”
The intestinal microecosystem maintains a dynamic equilibrium within the human body; however, external interventions or stimuli can disrupt this microbial homeostasis, leading to continuous changes as the system attempts to restore the original composition and structure of the gut microbiota. Evaluation of microbiome-related ecological indices can thus reflect the therapeutic efficacy of drugs in treating disease. [16-17] This study found that Scutellaria baicalensis extract can significantly restore the dysregulated composition of six bacterial phyla and 25 bacterial genera. The combined abundance of the phyla Bacteroides and Firmicutes accounts for more than 90% of the total bacterial population in the human body, according to Gulas et al. [18] Studies on the relative proportions of the phyla Firmicutes and Bacteroidetes have shown that when the ratio of Bacteroidetes to Firmicutes in intestinal contents exceeds the normal range, it accelerates the efficient absorption of dietary calories by gut microbes, thereby enhancing the rates of energy and metabolic processes in the body. The phylum Actinobacteria (primarily represented by bifidobacteria) and the phylum Proteobacteria (mainly comprising enterobacteria) are considered neutral microbial groups; however, if the intestinal microenvironment is disrupted, these groups can transform into invasive or pathogenic populations that migrate to other parts of the body, leading to pathological changes in the affected tissues or organ systems. [19] The results of this study demonstrate that Scutellaria baicalensis extract can modulate the abundance of Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria in a rat model of heat syndrome, thereby promoting the restoration of a disrupted intestinal microenvironment to its normal dynamic equilibrium and homeostasis. By taking the gut microbiota as an integrated whole, this study reflects the “holistic” principle underlying the therapeutic effects of the traditional Chinese medicine Scutellaria baicalensis in treating heat syndrome. The Spirochaetes and Fusobacteria are minor bacterial phyla within the host; with the continuous advancement of technologies in the biological sciences, their specific mycological significance remains to be further investigated.
Analysis of the associations between different bacterial genera and key indicators of the neuroendocrine–immune system revealed that the genus Collinsella exhibits strong correlations with the immune system, the central nervous system, and the endocrine system. Collinsella is a gas-producing genus that can promote the production of β-steroid dehydrogenase, accelerate the synthesis of ursodeoxycholic acid in the body, enhance the efficiency of metabolic conversion of substances and energy, and strengthen the body’s antioxidant capacity and immune function. [20-21] ; The genus Prevotella is a genus of non-spore-forming anaerobic bacteria belonging to the phylum Bacteroidetes, with acetate, succinate, and small amounts of butyrate as its major metabolic products. [22] , can promote the production of sphingolipid neurotransmitters and influence the feedback regulation of the immune and nervous systems. [23-24] The genus Paleobacter is an opportunistic pathogen with a strong propensity to infect immunocompromised individuals, potentially triggering excessive autoimmune responses and endocrine metabolic disturbances. [25-26] The findings of this study demonstrate that Scutellaria baicalensis extract exhibits significant modulatory effects on 25 differentially abundant bacterial genera and on key biomarkers of the neuroendocrine–immune axis, underscoring the multi-target, multi-pathway nature of traditional Chinese medicine and further highlighting its “holistic” therapeutic paradigm.
In summary, Scutellaria baicalensis extract can effectively ameliorate alterations in intestinal microbiota diversity and in the neuro–endocrine–immune axis in a rat model of heat syndrome. High-throughput 16S rRNA sequencing can be employed to establish an evaluation system for TCM syndromes, which holds profound guiding significance for research on relevant single herbs and compound formulations used to address such TCM syndromes. Changes in the gut microecology not only affect the diversity of the intestinal microbiota but also exert feedback effects on protein synthesis and transcription factors in relevant tissue cells. Moving forward, our research group will integrate multi-omics technologies with systems pharmacology approaches to provide a more in-depth elucidation of the scientific underpinnings of the TCM principle “cooling what is hot.”
Source: Li Zihui, Chen Pingping, Wang Yu, Zhang Yanan, Wu Juan, and Liu Shumin. Effects of Scutellaria baicalensis Extract on Gut Microbiota Diversity in a Heat-Excess Rat Model Based on High-Throughput Sequencing Technology [J]. Chinese Traditional Herbal Medicines, 2021, 52(2): 422–431.
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