Guiqi Baizhu Decoction Alleviates Radiation Inflammation in Rats by Modulating the Composition of the Gut Microbiota

The gut microbiota is important in metabolism and immune modulation, and compositional disruption of the gut microbiota population is closely associated with inflammation caused by ionizing radiation (IR). Guiqi Baizhu decoction (GQBZD) is a medicinal compound used in traditional Chinese medicine with anti-inflammatory and antioxidation effects, especially in the process of radiotherapy. However, the effect of GQBZD on reducing the damage to the normal immune system in radiotherapy remains unclear. Here, we show that GQBZD reduces body weights, water intake, food intake, diarrhea level and quality of life score, and inflammation and enhances immunity function in rats treated with X-ray radiation. Meanwhile, our data indicate that GQBZD not only reverses IR-induced gut dysbiosis as indicated change of α-diversity and β-diversity of microbiota, the composition of Desulfovibrio, Bacteroides, and Parabacteroides, except for Roseburia and Lachnoclostridium, but also maintains intestinal barrier integrity and promoting the formation of short-chain fatty acids (SCFAs). GQBZD can also reduce the level of phosphorylation P65 (p-P65). Our results demonstrate that GQBZD can significantly alleviate the inflammatory responses and improve the immune damage against IR, and may be used as prebiotic agents to prevent gut dysbiosis and radiation-related metabolic disorders in radiotherapy.


Introduction
Radiotherapy plays an important role in the treatment of both malignant and benign cancers for years [1]. However, its toxic effect to the surrounding normal healthy tissue remains a major drawback. Evidence has shown that the widespread use of ionizing radiation (IR) can cause high incidence of radiation-induced side effects in up to 75% during the process of radiotherapy [2]. e most common IR exposure damage effects are bone marrow inhibition and gastrointestinal toxicity [3,4]. Mechanisms of host damage caused by IR have been studied, and particularly, gut microbiota's responsiveness is a key part of IR [5]. It has been demonstrated that the gut microbiota modulates radiation therapy and chemotherapy efficacy through "TIMER" mechanisms, which indicates translocation, immunomodulation, metabolism, enzymatic degradation, and reduced diversity, but mechanism details of this response and the prevention are limited. e human gut microbiota consists of trillions of microbial cells and thousands of species of bacteria. Various internal and external environmental factors of the human body, including radiation, affect the balance of gut microbiota [6]. David et al. found that 16 O radiation induces multimodal responses in the mouse gut microbiome [7]. e microbiota diversity and composition were changed at the functional level, while the phosphatidylinositol signaling could be linked to dose-dependent changes in the abundance of specific taxa. Zhang and Steen also shows that gut microbiome is very sensitive to IR and can be used as radiation biomarkers [8]. e gut microbiota has increasingly been recognized as a key contributor to human health, and an imbalance in the colonic microbiota might underlie many human diseases, such as diarrhea, immune hypofunction, and inflammatory reaction of intestine. erefore, it is necessary to keep intestinal flora balance.
Most of the traditional Chinese medicine (TCM) produces effect by oral administration, which has good therapeutic effect against the illness. However, it is difficult to explain the detailed mechanism on TCM. Evidences have shown that TCM can alleviate diseases by regulating gut microbiota. Chang et al. indicate that G. lucidum and its high molecular weight polysaccharides can prevent gut dysbiosis and obesity-related metabolic disorders in obese individuals [9]. Alrafas et al. demonstrate that resveratrol modulates the gut microbiota to prevent murine colitis development through induction of Tregs and suppression of 17 cells [10]. e traditional Chinese herbal formula, Gegen Qinlian decoction (GQD), can alleviate type 2 diabetes and enrich the amounts of beneficial bacteria, such as Faecalibacterium spp. [11]. Chaihu-Shu-Gan-San (CSGS) formulas may be used as prebiotics to modulate gut microbiota structure and take the protective effects on the host metabolic phenotype of the gut microbiota dysbiosis rats [12]. Huangqin decoction (HQD) could ameliorate dextran sulphate sodium (DSS)-induced colitis through alteration of the gut microbiota [13].
Guiqi Baizhu decoction (GQBZD), an herbal medicine containing seven active compounds, consists of Astragalus membranaceus, Rhizoma Atractylodis Macrocephalae, Angelica, Paeonia lactiflora, dried tangerine peel, Rhubarb, and Licorice, has been widely used in China as traditional treatment for many diseases. is herb possesses a number of beneficial therapeutic properties such as cytoprotective, antioxidative, anti-inflammatory, and antitumor activities. Clinically, GQBZD was used to reduce proinflammatory factor expression and the release of serum tumor markers and improve the life quality of gastric cancer patients [14]. Wang et al. proved that GQBZD has an attenuated effect on cisplatin-induced kidney injury, and its mechanism may be related to improve blood activity and reduce apoptosis of kidney tissue cells [15]. However, it is still unknown whether GQBZF can prevent radiation and what is its detail mechanism. In this study, using the high-throughput 16S rRNA gene sequencing, we identified specific fecal microbial signatures in rats with radiation and sought to elucidate potential biomarkers or mechanistic principle show that the gut microbiota dysbiosis may impact the pathogenesis of radiation, and to investigate the protective effect of GQBZD on radiation-induced enteritis and its relationship with gut microbiota. e results maybe provide useful information about the therapeutic value of microecological preparation for radiation.

Materials and Methods
2.1. Animal. SD rats (6 weeks of age to 8 weeks of age, thirty males and thirty females) were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. e animal certificate number is 62000600000293. All animal experiments reported in this study were approved by the Ethics Committee of Gansu University of Chinese Medicine. All animals housed in a controlled room (temperature, 25 ± 2°C; relative humidity, 45-60%; lighting cycle, 12 h/d; 06:30-18:30 for light) and had free access to food and drinking water during the experimentation.
e dose rate was about 200 cGy/min (225.0 kV, 13.3 mA). e total absorbed dose was 6 Gy. All irradiations were performed at room temperature.
Five samples were randomly selected, and high-performance liquid chromatography (HPLC) was used to detect the level of major components in the abovementioned GQBZD per dose, named Astragaloside IV, Paeoniflorin, and Hesperidin. HPLC analyses were performed on a Shimadzu liquid chromatography system (Shimadzu LC-2030, Kyoto, Japan) equipped with an evaporative light detector (Shimadzu 228-4511538, Kyoto, Japan). e chromatographic separation of the analytes was performed on a SinoChrom ODS-BP C18 analytical column (LC Column, 250 mm × 4.6 mm, 5 μm). e sample injection volume was 10 μL. An evaporative light-scattering detector was used to detect Astragaloside IV, and an ultraviolet detector was used to detect Paeoniflorin and Hesperidin. e wavelength of the Astragaloside IV, Paeoniflorin, and Hesperidin detectors was

Groups.
Sixty SD rats were randomly assigned into Ctrl, Drug, IR, LD, MD, and HD groups (n � 10 each group). Rats in the Ctrl group were lavaged daily with saline. Rats in the IR group were treated with 6 Gy of X-ray irradiation and lavaged daily with saline. Rats in the LD group were treated with 6 Gy of X-ray irradiation and lavaged daily with low dose of GQBZD (4.1 g/kg). Rats in the MD group were treated with 6 Gy of X-ray irradiation and lavaged daily with middle dose of GQBZD (8.2 g/kg). Rats in the HD group were treated with 6 Gy of X-ray irradiation and lavaged daily with high dose of GQBZD (16.4 g/kg). Rats in the Drug group were treated similarly to the MD group (8.2 g/kg) but without irradiation. Rats were lavaged daily with saline and GQBZD at corresponding doses, respectively, for seven consecutive days.

Hematoxylin and Eosin (HE) Staining.
Colon tissues were fixed with 4% paraformaldehyde at room temperature for 24 hours. e samples were embedded in paraffin, dehydrated, then sectioned at a 5 μm thickness. e sections were stained with hematoxylin and 0.5% eosin for 5 and 3 minutes, respectively. Tissue sections were observed under a light microscope. Biological verifications were performed six times in each group.

ELISA Test.
e expression of cytokines and inflammatory-associated proteins including IL-4, IL-6, IL-10, TNF-α, sIgA, PKC, HSP, ALP, and DAO in each group at different time points was detected by the ELISA test. e specific operation method was carried out following the manufacturer's instructions. Biological verifications was performed six times in each group.

Microbiota
Profiling. Before the mice were sacrificed, the mice were fixed and their tails were lifted. We gently pressed the mouse's lower abdomen with our fingers and collected fresh feces in several sterile EP tubes with corresponding numbers. e EP tubes were stored at −80°C and transported with dry ice.
Total genome DNA from fecal samples was extracted for amplification using specific primer with the barcode (16S V3 + V4). Paired end sequencing was performed on the Illumina MiSeq Platform, and a phylogenic tree and OTU table were obtained from the Mothur Bayesian classifier. Sequencing libraries were generated and analyzed according to Zhang et al.'s previous study [16]. Principal coordinate analysis (PCoA) was performed to get principal coordinates and visualize from complex, multidimensional data. Observed-species, Shannon, Simpson, Chao1, and ACE are used to evaluate the complexity of species diversity [17]. OTUs were further used for genome prediction of microbial communities by PICRUST [18]. Biological verifications were performed six times in each group (three females and three males in each group).

Statistical Analysis.
All statistical analyses were performed by using the one-way analysis of variance (ANOVA) to test homogeneity of variances via Levene's test and followed with Student's t-test (SPSS 21.0 software). Data are expressed as the mean ± SEM and denoted as follows: ns P > 0.05; * P < 0.05; * * P < 0.01; * * * P < 0.001.

e Contents of Main Components in GQBZD.
e purposes of detecting the contents of the main components in GQBZD was to ensure the uniformity and standardization of the drugs. As shown in Figure 1, HPLC analyses revealed that the content of Astragaloside IV was 68.69 ± 3.94 mg/kg, the content of Paeoniflorin was 2127.43 ± 17.78 mg/kg, and the content of Hesperidin was 273.54 ± 9.73 mg/kg.

GQBZD Enhances the Life Quality and Immune Function of the Rats Exposed to IR.
To determine the effect of GQBZD on X-ray radiation rats, body weight, water intake, food intake, quality of life score, diarrhea level score, and the immune function were tested. As shown, X-ray radiation significantly decreased the body weight, water intake, food intake, quality of life score, and diarrhea level score (P < 0.05) (Figures 2(a)-2(e)) and X-ray radiation significantly decreased the nucleated cells in bone marrow, white blood cell in blood, splenic index, and the secretion of sIgA both in serum and in colon (P < 0.05) (Figures 3(a)-3(f )). GQBZD treatment tended to alleviate X-ray radiation-induced weight loss, water intake loss, and food intake loss (P < 0.05) (Figures 2(a)-2(c)), and GQBZD treatment alleviated the life quality and diarrhea level scores in X-ray radiation rats (Figures 2(d)-2(e)). In addition, GQBZD treatment alleviated X-ray radiation-induced immune function. e nucleated cells in bone marrow, white blood cell in blood, splenic index, and the secretion of sIgA in serum and in colon increased in the GQBZD group compared to the X-ray radiation group (P < 0.05) (Figures 3(a)-3(f )), whereas GQBZD enhances the life quality and immune function in rats treated by X-ray radiation.   Figure 2: Effect of GQBZD on the state of affairs to rats in X-ray radiation. e results depict body weight (a), water intake (b), food intake (c), quality of life score (d), and diarrhea level score (e). Values are presented as mean ± SEM. Differences were assessed by ANOVA for multiple comparisons and denoted as follows: * P < 0.05; * * P < 0.01; * * * P < 0.001; ns P > 0.05.

GQBZD Alleviates the Inflammatory Response to X-Ray Radiation.
To determine the effect of GQBZD on the immune system, the expression of inflammatory cytokines were detected. e results showed that X-ray radiation significantly decreased the secretion of IL-4, IL-10, HSP, PKC, and ALP and increased the expression of IL-6, TNF-α, and DAO (P < 0.05) (Figures 4(a)-4(j)), whereas GQBZD treatment changed the secretion of IL-4, IL-10, HSP, PKC, and ALP increased, and the expression of IL-6, TNF-α, and DAO decreased in GQBZD compared to the X-ray radiation group (P < 0.05), indicating that GQBZD treatment alleviates X-ray radiation-induced inflammatory response (Figures 4(a)-4(j)).

GQBZD Attenuates Radiation-Induced Colitis and Intestinal Damage.
To determine the effect of GQBZD on the inflammatory injury of intestinal in X-ray radiation rats, HE stain of colon, protein Occludin, ZO-1, P65, and p-P65 were

GQBZD Reprograms the Gut Microbiota in Radiation
Rats. Gut microbiota is highly associated with inflammation; thus, we further determined fecal microbiota compositions by sequencing the bacterial 16S rRNA (n � 6). Shannon, Simpson, and ACE indexes were examined for the alpha-diversity of the microbiomes. Radiation rats exhibited a lower diversity of microbiota evidenced by the decreased Shannon, Simpson, and ACE indexes compared to controls (P < 0.05) (Figures 6(a)-6(c)). Interestingly, GQBZD treatment markedly increased Shannon and Simpson indexes (P < 0.01), suggesting an improvement in gut microbiota diversity in radiation rats. To evaluate overall differences in beta-diversity, we applied PCoA plot and PCA space diagram to weighted and unweighted UniFrac distance metric matrices generated for the sample set. Control and radiation rats presented a distinct clustering of microbial community structure, while the GQBZD group had a similar structure to that of the control rats (Figures 6(d)-6(e)). e overall microbial composition in each group differed at the phylum, class, order, family, and genus levels (Figures 7(a)-7(e)). e largest phylum represented in each dataset was Firmicutes, Bacteroidetes, and Proteobacteria. Radiation rats had a higher relative abundance of Bacteroidetes and Proteobacteria, but a lower relative abundance of Firmicutes and the ratio of Firmicutes/Bacteroidetes (P < 0.001), while GQBZD markedly reversed these alterations in Firmicutes, Bacteroidetes, and Proteobacteria and the ratio of Firmicutes/Bacteroidetes (Figures 7(f )-7(h)). At the genus level, the top 10 genera represented in each dataset were Phascolarctobacterium, Oscillospira, CF231, Bacteroides, Lactobacillus, Dorea, Parabacteroides, Ruminococcus, Prevotella, and Desulfovibrio. Rats treated with IR had a higher relative abundance of Oscillospira, CF231, Bacteroides, and Parabacteroides (P < 0.05), and a lower relative abundance of Phascolarctobacterium, Lactobacillus, Dorea, and Prevotella (P < 0.05), but changed not in Desulfovibrio (P > 0.05). Fortunately, GQBZD could change most of the above indicators and it markedly reversed these alterations in Phascolarctobacterium, Oscillospira, CF231, Bacteroides, Dorea, Parabacteroides, and Prevotella (P < 0.05) but except in Lactobacillus (P > 0.05). An interesting thing was that Desulfovibrio responses to GQBZD but not IR, and it raised by the action of GQBZD drugs only (Figures 6(i)-6(q)).

Discussion
Ionizing radiation (IR), a recognized proinflammatory agent, can cause oxidative stress, inflammation, p53 induction, DNA damage, mutagenesis, and oxidation of various molecules such as lipids and proteins [19][20][21]. IL-4, IL-6, IL-10, and TNF-α are representative inflammatory factors, PKC, HSP, ALP and DAO are products of stress reaction, and both of them are all sensitive to radiation and have response to radiation stress [21][22][23]. e current study demonstrated that IR (6 Gy) decreased the body weight gain, the number of nucleated cells in bone marrow, spleen index, and the screen of sIgA both in colon and serum, indicating immune function descent. Meanwhile, in this paper, X-ray radiation significantly decreased the anti-inflammatory factor secretion of IL-4 and IL-10, and increased the proinflammatory factor expression of IL-6, TNF-α, HSP, and PKC, whereas IR promoted the inflammation and alleviated immune function in rats.
is is the same as Mukherjee et al.'s study [24]. GQBZD is a traditional Chinese Medicine. Studies from our laboratory and in clinic   Evidence-Based Complementary and Alternative Medicine have shown its ability to reduce the symptoms associated with colitis in mice and crowd models, and GQBZD conferred meaningful prevention on inflammation in rats [14,15]. It has been reported that gut microbiota plays a great role in the course of action of traditional Chinese medicine [25]. e anti-inflammatory, radio-resistance effects of TCM may be partly mediated by regulating the composition of gut microbes [26]. Many effective components of GQBZD have been proved to have antiradiation effect. Li et al. demonstrated that Astragaloside IV showed radio protective effects on the hematopoietic system in mice [27], and Liu et al. suggest that Astragaloside IV can antagonize radiation-induced brain cells senescence, and its mechanism may be related to p53-p21 and p16-RB signaling pathways of aging regulation [28]. Yue et al. found that Paeoniflorin enhances its sensitivity to radiotherapy by regulating MLH1 and MSH1 protein to enhance the DNA damage and repair ability of melanoma cells [29]. Santhanam et al. obtained that Hesperidin has the potential to be used as active ingredients in sunscreen and antiphoto aging formulations [30]. Hewage et al. found that Hesperidin shielded human keratinocytes from UVB radiation-induced damage and apoptosis via its antioxidant and UVB absorption properties [31]. In clinic, we also have observed that GQBZD is a safe and effective treatment for diarrhea of gastric cancer patients and that it can slightly alleviate diarrhea associated with irritable bowel syndrome in tumor. We, therefore, speculated that GQBZD's effects on the development of radiation might depend on gut microbiota. Diarrhea is among the most common side effects of radiation therapy for malignant tumor [27,32,33]. ere is evidence to suggest that changes in the gut microbial balance are involved in the onset of radiation-induced diarrhea: probiotics have been shown to prevent occurrence of radiation-induced diarrhea, and germ-free mice are more resistant to radiation enteritis [34]. In this study, we took advantage of the SD rat model of radiation that allowed localized injury to the rectum by abdominal irradiation which mimics human radiation proctitis. We discovered a significant α-diversity declining and β-diversity changing in colon contents collected from the radiation rats. Many research shows this is a common phenomenon in multiple animal models, such as in Bactrocera dorsalis [35], in the bank vole [36], in the Gottingen minipig and rhesus macaque models [37], and in mice and human [6,7]. Besides, similar to the study of Casero et al. [7], a significant increase was revealed in the relative abundance of Bacteroidetes and Proteobacteria and decrease was revealed in the relative abundance of Firmicutes at phylum level in the IR rats, and GQBZD could reverse the changes. Dysbiosis of gut microbiota was typified by a bloom in the ratio of Bacteroidetes and Firmicutes. In this study, radiation rats had a higher relative abundance of Bacteroidetes and Proteobacteria, but a lower relative abundance of Firmicutes and the ratio of Firmicutes/Bacteroidetes, while GQBZD markedly reversed these alterations in Firmicutes, Bacteroidetes, Proteobacteria and the ratio of Firmicutes/Bacteroidetes. e largest genus represented in each dataset were Desulfovibrio, Bacteroides, Parabacteroides, Lactobacillus, Roseburia, and Lachnoclostridium. Radiation rats had a higher relative abundance of Desulfovibrio, Bacteroides, Parabacteroides but a lower relative abundance of Lactobacillus, Roseburia, Lachnoclostridium (P < 0.001), while GQBZD markedly reversed these alterations in Desulfovibrio, Bacteroides, Parabacteroides, and Lactobacillus (P < 0.01) but except in Roseburia and Lachnoclostridium.
Variation of the composition of intestinal flora is often accompanied with the changes of the function of intestinal flora and the difference to the metabolic production of intestinal flora. In this paper, the PICRUST analysis was used to predict the gut microbiota function and the metabolites. e results show that radiation markedly reduced the level of anaerobic, the function of stress-tolerant, and contains-mobile-elements decreased, but enhanced the level of aerobic and the possibility of potentially-pathogenic. GQBZD reprogramed the expression of them. e short-chain fatty acids (SCFAs), metabolites of gut microbial, have shown promise for treating inflammatory bowel disease [38], mediating intestinal barrier function [39] and increasing anti-inflammatory cytokine secretion [40]. In this paper, we take advantage of gas chromatography-mass spectrometry to test the expression of acetic acid, propionic acid, and butyric acid, important metabolites of the intestinal flora. e result demonstrated that radiation markedly reduced the level of acetic acid, propionic acid, and butyric acid, and GQBZD treatment reprogramed them. It has been reported that species of Desulfovibrio has the ability to reduce several toxic metals such as uranium, chromium, and iron [41], Desulfovibrio has the potential to generate energy (ATP) through electron transfer-coupled phosphorylation [42], and Bacteroides is the producer of butyric acid, induces IL-10 secreting B-and T-cells that prevent viral encephalitis [43]. It is an evidence that variation of the composition of the gut microbiota accompanied with the alteration of the function and the metabolic production of the gut microbiota, and GQBZD can partially relieve the changes of intestinal microecological components.
Intestinal homeostasis is maintained by a unique underlying inflammatory tone that is modulated by many pathways. Disorders of these pathways are associated with an increase in intestinal damage. Studies have shown that NF-κB signaling in epithelium is an important homeostatic pathway regulating intestinal inflammatory response [44]. Intestinal epithelial specific conditions in knockout mice suggest that normalizing the NF-κB pathway is essential for the survival and protection of colitis epithelial cells [45]. Interestingly, activation of the constitutive NF-κB pathway may also lead to increased expression and injury of inflammatory mediators in intestinal epithelial cells [46]. In this work, we tested the level of P65 and the phosphorylation P65. e phosphorylation sites of P65 are located in the S276 regions of the N-terminal active region. When serine 276 is phosphorylated, P65 can be activated and the inflammation levels in the downstream increased in the IR group. GQBZD can downregulate the P65 phosphorylation. Our findings indicated that the potential applications of GQBZD in the treatment of radiation-induced systemic inflammation and metabolic syndrome might closely depend on its modulatory effect on gut microbiota. To confirm the results of GQBZD acting on gut microbiota, a more complete analysis of the microorganisms in the gut is suggested for further studies.

Conclusion
In this study, a proposed model of regulating intestinal microecology by GQBZD in rats was summarized (Figure 9). e NF-κB signaling pathway is probably one of the mechanisms underlying the regulating intestinal microecology of GQBZD. In summary, rats treated with IR experienced change with the composition of the gut microbiota, and its metabolites and biological functions changed.
is maybe leads to intestinal permeability increased and inflammatory level increased and immune decreased. Our results disclose that GQBZD alleviates radiation inflammation in rats by modulating the composition of the gut microbiota and suppresses P65 activation, as well as decreases the expression of the inflammatory factor. In this work, gut microbiota may be the target of GQBZD, but "who" GQBZ works for and "how" GQBZD works is an interesting topic worth further investigation.

Data Availability
Due to confidentiality agreements, supporting data can only be made available to bona fide researchers subject to a nondisclosure agreement. Details of the data and how to request access are available from data manager contact info at the Provincial-Level Key Laboratory for Molecular Medicine of Major Diseases and e Prevention and Treatment with Traditional Chinese Medicine Research in Gansu University of Chinese Medicine.  Evidence-Based Complementary and Alternative Medicine 11