LIU He,FAN Qing-Jie,LIU Yin-Hui,LI Ming,YUAN Jie-Li

College of Basic Medical Science,Dalian Medical University,Dalian,Liaoning 116044,China

Keywords

Ganoderma lucidum

Bacillus subtilis

Traditional Chinese medicine fermentation

Ganoderma lucidum acid

Intestinal mucosal barrier function

Intestinal dysbiosis

Regulate T cell responses

ABSTRACT

Objective This study aimed to develop a type of Ganoderma lucidum(G.lucidum)-probiotic fermentation broth that can effectively improve the intestinal mucosal barrier function of mice with ceftriaxone-induced intestinal dysbiosis.

Methods By means of absorbance of optical density(OD)value and phenol-concentrated sulfuric acid measurement of polysaccharide content,the probiotic species can grow on the medium of G.lucidum were screened out,and the concentration of the medium of G.lucidum was determined,and the fermentation broth was prepared for subsequent experiments.Thirty-two SPF grade male BALB/c mice were randomly divided into four groups(eight mice in each group),namely control group(CON),intestinal mucosal barrier damage model group(CS),fermentation broth intervention group(FT)and G.lucidum medium intervention group(GL)，respectively.The intestinal dysregulation model was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(twice a day for seven consecutive days).From day 8,the FT group and GL group were gavage with 0.2 mL fermentation broth and G.lucidum medium,respectively.On day 15,all mice were sacrificed.To draw the weight curve and measure the cecal index; pathological examination of colon tissues with HE staining; serum levels of LPS,IL-10,TNF and IL-6 were detected by ELISA.Flow cytometry was used to analyze the changes of CD3 + T cells,CD4 + T cells,CD8 + T cells and macrophages in spleen.16S rRNA sequencing was performed to detect the intestinal microbiota structure of mice.

Results Bacillus subtilis can decompose and utilize G.lucidum fruiting body medium,and the suitable concentration of G.lucidum fruiting body medium is 33.2 mg/mL.The effect of Bacillus subtilis-G.lucidum fermentation broth on the damage of intestinal mucosal barrier caused by ceftriaxone sodium was reduced,the body weight of mice recovered and colon swelling improved,colon histopathological injury was alleviated,inflammatory cell infiltration was alleviated,serum IL-10 increased significantly,LPS、TNF-αand IL-6 decreased significantly compared with model group,and the proportion of T cells and intestinal dysbiosis was improved.

Conclusions The experimental results suggest that Bacillus subtilis-G.lucidum fermentation broth can effectively improve the intestinal barrier function damage,immune dysfunction and intestinal dysbiosis caused by antibiotic overdose,and has a certain regulatory effect on intestinal mucosal barrier function.

1 Introduction

Ganoderma lucidum(G.lucidum)is widely distributed in China and modern medical research has shown that it has a variety of pharmacological effects,such as anti-inflammatory[1]and anti-tumor activities[2],immunity enhancement[3],and delayed aging,and has exhibited varying degrees of regulatory improvement in the cardiovascular system,nervous system and respiratory system[4].The chemical composition ofG.lucidumis complex,and at present,the main known constituents areG.lucidumpolysaccharides(GLP),G.lucidumtriterpenoids(GLT),alkaloids,nucleosides,amino acids,organic germanium,fatty acids,and various trace elements and inorganic ions[5].GLP can regulate cellular immunity[6]and intestinal mucosal immunity[7]and indirectly kill tumor cells by enhancing the functions of macrophages,NK cells and cytotoxic T cells[8,9].GLP can also kill tumor cells or promote their apoptosis via the release of cytokines such as tumor necrosis factor and interferons by macrophages and T cells[10].The diversity of chemical structures in ganosporic acid(generally known as GLT)gives it a wide range of pharmacological effects,such as anti-tumor,antimicrobial,anti-hypertension activities[11],anti-HIV-1 and HIV-l protease activities[12],inhibition of angiotensin converting enzyme[13],inhibition of platelet aggregation as well as antioxidant effects[14].However,as macromolecules,how these GLPs and GLTs exert their effects in the human body remains unclear.

One hypothesis is that the human gut microbiota utilize theG.lucidumcomponents and in turn affect the intestinal mucosal barrier functions as well as the local and systemic immune responses.The normal human gastrointestinal tract is inhabited by a large number of microorganisms,which are collectively referred to as the intestinal microbiota.Normally,there are an estimated 100 trillion bacteria in an adult human body(approximately 10 times the number of human cells),and 80% of these bacteria are found in the gut[15].The intestinal microbiota maintains a dynamic balance through co-metabolism with the host.Once affected by changes in host and external environments,especially due to long-term use of broadspectrum antibiotics,sensitive intestinal bacteria are inhibited and the number of uninhibited bacteria increases,resulting in bacterial dysbiosis[16].Intestinal dysbiosis has been found to be closely related with diseases of the digestive system,endocrine system,central nervous system and metabolic system[17].The intestinal microbiota plays an important role in intestinal barrier function by providing intestinal mucosal cells with certain nutrients and maintaining the balance of the intestinal microecosystem,as well as activating the intestinal immune system.In addition,the gut microbiota has a strong ability to utilize a variety of natural polysaccharides,such as GLP and convert these into beneficial metabolites,such as short-chain fatty acids.However,when dysbiosis occurs in the gut of a patient,this conversion may be disturbed due to the reduced abundance of probiotic bacteria such asLactobacillusandBifidobacteria.Therefore,the development of better interventions is urgently needed.

Fermentation of traditional Chinese medicine(TCM)refers to the decomposition reaction of the treated medicinal materials or extracts of medicinal materials by microorganisms under specific temperature and humidity conditions.The fermentation process of probiotics in Chinese herbal medicines can improve the content of active compounds,and the pre-digestion of probiotics can further reduce the toxicity and irritation of Chinese herbal medicines,thus promoting the absorption and utilization of the active compounds.Modern Chinese medicine fermentation technology has been widely used in food,medicine and other industries[18,19].Therefore,in this study,we investigated whether the probiotic-G.lucidumfermentation broth could ameliorate the intestinal dysbiosis and intestinal barrier damage caused by ceftriaxone in mice.

2 Materials and Methods

2.1 Selection of probiotic strains that ferment G.lucidum

An appropriate amount of fruity powder ofG.lucidumwas added to the liquid MRS medium(content based on TCM formula),which was then sterilized for 20 min at 121 °C and high pressure,followed by filtration through an aseptic filter.The ability of bacterial strains to grow inG.lucidum-MRS medium was assessed by measuring the absorbance of the culture broth at OD 620 nm after cultivation at 37 °C for 48 h.The bacterial strains used in this study include:Bacillus subtilis(CGMCC 1.157 92),Lactobacillus brevis(CGMCC 1.202 8),Bifidobacterium breve(CGMCC 1.300 1),Lactobacillus casei(CGMCC 1.872 7),Bifi dobacterium pseudocatenulatum(CGMCC 1.500 2)andLactobacillus gasseri(CGMCC 1.339 6).In order to select the best condition for fermentation ofG.lucidumby probiotic strains,we adjusted the concentration of the preparedG.lucidum-MRS medium to three concentrations:low(8.8 mg/mL),medium(16.6 mg/mL)and high(33.2 mg/mL).

2.2 Detection of the fermentation products of G.lucidum by Bacillus subtilis

To test theG.lucidumfermentation rate of the probiotics,we analyzed the polysaccharide content in the fermentation broth by applying the phenolsulfuric acid method[20].Afterwards,the detailed composition of theG.lucidum-Bacillus subtilisfermentation broth was determined by high performance liquid chromatography(HPLC)and compared with theG.lucidum-MRS medium.The instruments used in the HPLC were a Shimadzu ultra-HPLC AB SCI EXX500 series QTOF mass spectrometer,SPE solid phase extraction device,nitrogen blowing instrument(MTN-2800D),and a vortex mixing machine(VORTEX2).The chromatographic column was a Waters ACQUITY UPLC HSS T3(100 mm×2.1 mm,1.8 μm),in which the mobile phases A and B were water and 1% formic acid in water,respectively; the flow rate was 0.4 mL/min,and the column temperature was 30 °C.At the same time,we measured the pH of the probiotic-G.lucidumfermentation broth and theG.lucidummedium.

2.3 Mice

For the animal experiment,32 male BALB/c mice(aged 6 - 8 weeks,weighing 18 - 22 g)were obtained from the specific pathogen free(SPF)from the Experimental Animal Center of Dalian Medical University,China.The procedure for the animal experiments was approved by the medical ethics committee of Dalian Medical University,China.The animal certificate number is AEE17013.

2.4 Induction of intestinal dysbiosis in mice by ceftriaxone and interventions

Thirty-two mice were randomly divided into four experimental groups(n=8 for each group).The intestinal dysbiosis model was induced by intragastric administration of 0.2 mL ceftriaxone sodium(400 mg/mL,Shandong,China)to each mouse,twice a day for 7 d.In the control group(CON),the mice received physiological saline of equal volumes for 7 d,instead of ceftriaxone sodium.From day 8,the fermentation broth(FT)group and theG.lucidumbroth(GL)group were administered the probiotic-G.lucidumfermentation broth and theG.lucidummedium 0.2 mL,respectively,after the intestinal barrier imbalance model was completed.As a control,the ceftriaxone sodium(CS)group was given saline solution.Body weight was measured at the same time every day.On day 15,all mice were sacrificed by cervical dislocation,and stool samples collected from the mice were stored at - 80 °C before analysis.The length of the colon and the weight of the cecum were measured,and the ratio of the weight of the cecum to body weight was calculated.

2.5 Histological evaluation of intestinal mucosa

After the mice were sacrificed,the colon of each mouse was excised,and the adherent adipose tissue was removed.The colon specimens were fixed in 4%paraformaldehyde,embedded in paraffin sections(5 μm),and stained with hematoxylin and eosin(HE).The tissues were scored based on two histological characteristics(the extent of cell infiltration and tissue damage),which ranged from 0 to 6.

2.6 Measurement of serum lipopolysaccharides(LPS)and cytokine levels

Blood samples were collected and centrifuged at 986 g for 15 min at 4 °C to obtain serum samples.The concentration of serum LPS was measured using ELISA kits(Shanghai Lengton Bioscience Co.,Ltd.,Shanghai,China).Tumor necrosis factor(TNF)-α,interleukin(IL)-10 and IL-6 were detected by enzymatic methods according to the instructions of the relevant assay kits(Shanghai Lengton Bioscience Co.,Ltd.,Shanghai,China).Absorbance measurement was carried on a computer interface microplate reader(BioRad,Houston,TX)at 450 nm,and the results were expressed as ng/L,pg/mL and pg/mL respectively.

2.7 Flow cytometric analysis

The fresh spleen of each of the euthanized mice was removed and gently ground in PBS buffer,to obtain approximately 1×106cells as needed for flow cytometry(FACS).Cells were incubated with anti-mouse CD16/CD32 mAb to block Fcγreceptors for 60 min and then stained on ice for 60 min with combinations of monoclonal-antibodies.The mAbs used in this study were anti-mouse CD16/CD32(2.4G2),PElabeled anti-mouse CD3(2C11),APC-labeled antimouse CD4(53-6.7),TITC-labeled anti-mouse CD8(LT8),and PE-labeled anti-mouse F4/80(BM8),which were all purchased from BD Biosciences.The flow cytometry was run on an FACS-Calibur(Becton Dickinson,Mountain View,CA,USA),and the results were analyzed using FlowJo software(Tree Star).

2.8 Fecal DNA extraction,PCR and 16S rRNA gene sequence analysis

The metagenomic microbial DNA was extracted from fecal samples of the mice in each group using the stool DNA kit(Omega Bio-Tek,Inc.)according to the manufacturer’s instructions.The v3-v4 region of bacterial 16S rRNA gene was amplified by polymerase chain reaction(PCR)with a forward primer(5′-ACTCCTACGGGAGGCAGCA-3′)and a reverse primer(5′-ATTACCGCGGCTGCTGG-3′).The PCR reaction system(50 μL)included the following:5 μL cDNA,25 μL PCR 2×Easy Taq Super mix(HotStar-Taq Plus DNA polymerase,dNTPs,MgCl2 and reaction buffer),1 μL each of the forward and reverse primers,and 18 μL deionized water.The PCR reaction was started with 95 °C denaturation and lasted 2 min,30 cycles consisting of:94 °C for 45 s,55 °C for 45 s,and 72 °C for 60 s,at which point the 72 °C incubation was terminated and cooled for 8 min at 4 °C.We sequenced the PCR amplicons and analyzed the data using Illumina HiSeq(Novogene,Beijing,China).

2.9 Statistical analysis

The data are presented as means±SD.For the statistical analysis of quantitative multiple group comparison,one-way analysis of variance(and nonparameter)was performed,followed by Tukey analysis(comparing all column pairs).GraphPad Prism 5(Graph Pad Software,La Jolla,CA,USA)was used to conduct the nonparametricttest for comparison between two groups.P＜0.05 were considered statistically significant.

3 Results

3.1 Bacillus subtilis can grow in G.lucidum-MRS medium

The different strains of probiotics were inoculated into theG.lucidum-MRAS medium at 1%(v/v)concentration.After cultivation for 48 h in a shaker incubator at 37 °C,the optical density(OD)was measured at 620 nm to detect the growth of different bacterial strains and investigate which probiotic grows best withG.lucidumsupplementation.The growth curves of six probiotic strains used in this study are shown in Figure 1A.Each of the tested strains showed a certain trend of utilizingG.lucidum.In particular,the growth ofBacillus subtiliswas clearly improved in theG.lucidum-MRS medium when compared with the control group and the other treatment groups.In order to determine the appropriate concentration ofG.lucidum-MRS medium,we prepared low(8.8 mg/mL),medium(16.6 mg/mL)and high(33.2 mg/mL)concentration groups and inoculated these with theBacillus subtilis.We found that the optical density(indicating bacterial growth)was highest when the concentration ofG.lucidumwas 33.2 mg/mL and the polysaccharide content was significantly higher than that of the control group at each time period(Figure 1B and 1C),which suggests an efficient utilization ofG.lucidumpolyglycans byBacillus subtilis.

3.2 Changes in the composition of G.lucidum after fermentation by Bacillus subtilis

To investigate the features of the probiotic-G.lucidumfermentation broth,we used HPLC to compare the differences in the main composition ofG.lucidumbefore and afterBacillus subtilisfermentation.The results showed a significant difference in composition between the fermentation broth and theG.lucidum-MRS medium itself.The abundance of a variety ofG.lucidumtriterpenoids,such as lucidenic acid N,ganoderic acid AM1 and ganoderenic acid B,was greatly reduced,which suggests consumption byBacillus subtilis(Figure 2A).The comparison of the response values of the components of probiotic-G.lucidumfermentation broth andG.lucidum-MRS medium is shown in Figure 2B.As well,it was found that the pH value of the fermentation broth was significantly lower than that of theG.lucidum-MRS medium,which indicates thatBacillus subtiliscould transformG.lucidumtriterpenoids into organic acids(P＜0.000 1,Figure 2C).

3.3 G.lucidum-Bacillus subtilis fermentation broth ameliorated the weight loss and cecum swelling induced by ceftriaxone sodium

The mouse model of intestinal dysbiosis was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(400 mg/mL)for 7 consecutive days(Figure 3A).Significant weight loss was observed in the ceftriaxone sodium-treated mice(the CS group)compared with the CON group.Administration ofG.lucidum-MRS medium(GL)andG.lucidum-Bacillus subtilisfermentation broth(FT)to the model mice significantly ameliorated the weight loss at 7 - 14 d(P＜0.000 1,Figure 3B).Compared with the GL group,the body weight of mice in the FT group increased more rapidly after day 11,suggesting a better recovery.The cecum index(the ratio of cecum weight to body weight)is another indicator of the severity of intestinal inflammation.Although the cecum index increased noticeably after ceftriaxone sodium intake,amelioration was evident(indicated by the appearance of the cecum)in mice in both TF and GL groups; however,the cecum index showed no statistically significant change in either of the treatment groups compared with the CS group(Figure 3B and 3C).

3.4 G.lucidum-Bacillus subtilisfermentation broth mitigated the histological damage of the colon and reduced the intestinal permeability

The colorectal section with HE staining showed that the mice that had been given ceftriaxone sodium without any other treatment(the CS group)suffered severe damage,including distortion of crypts,thickening of the muscularis externa,shedding of goblet cells,and the apparent infiltration of inflammatory cells in the mucosa and submucosa.TheG.lucidum-Bacillus subtilisfermentation broth had a clear protective effect on the colon recess structure,and histological inflammation was light.The histological protection of the colon in the GL group was not as pronounced as that in the FT group colons,in which there was still infiltration of a large number of inflammatory cells and distortion of crypts(Figure 4A and 4B).In addition,the fermentation broth significantly reduced the levels of lipopolysaccharides(LPS)in the FT group(P=0.002 5),and the LPS level in the GL group was significantly lower than that of the CS group(P＜0.000 1).However,there was no significant difference in LPS level between the GL group and the FT group,which is an indication of reduced intestinal permeability(Figure 4C).The data suggest that probiotic-G.lucidumfermentation improved the mucosal barrier function in ceftriaxonetreated mice.

3.5 G.lucidum-Bacillus subtilisfermentation broth showed superior effect in improving the secretion of anti-inflammatory cytokines

To investigate the anti-inflammatory effect of the fermentation broth,serum IL-10,TNF-αand IL-6 levels were measured using ELISA.After administration of ceftriaxone sodium,the serum level of IL-10 in the mice was reduced(P=0.031 4).The fermentation broth treatment(FT),as well as theG.lucidum(GL),significantly increased the level of IL-10 in the serum(P＜0.000 1,P=0.000 5,Figure 5A),and no statistical difference was found between GL and FT groups.As shown in Figure 5B,the level of TNF-αin FT and GL groups was significantly lower than that in CS group(P=0.042 5,P=0.000 3,respectively),and the TNF-αlevel of FT and GL groups were close to the CON group.The TNF-αlevel in the GL group was lower than that of the FT group,and this difference was statistically significant(P=0.041 3).As shown in Figure 5C,the level of IL-6 in FT and GL groups was significantly decreased compared with that of the CS group(P=0.002 9,P=0.000 4).The IL-6 level in the GL group was lower than that of the FT group,and there was a statistically significant difference(P=0.007 5).These results suggest that FT and GL can relieve the chronic systemic inflammation caused by ceftriaxone sodium in mice.

3.6 G.lucidum-Bacillus subtilis fermentation broth can regulate T cell responses in ceftriaxone-treated mice

To further investigate whether theG.lucidum-Bacillus subtilisfermentation broth could affect the immune responses of ceftriaxone-treated mice,we examined the proportions of lymphocytes in the mice spleens.Flow cytometry analysis showed that the number of mature T cells(CD3+)increased significantly in the spleens of the CS group(P=0.04),whereas it markedly decreased in the FT group(P=0.023 5,Figure 6A).The level of CD4+T cells in the CS group was higher than that in the CON group,but there was no statistically significant difference.CD4+T cells in the FT group were markedly lower than those in the CS group(P=0.030 7,Figure 6B).Similarly,the proportion of CD8+T cells also increased in the ceftriaxone-treated mice(P=0.000 4)and decreased after FT treatment(P=0.002 1),and the level of CD8+T cells was close to the CON group; however,no improvement was found when mice were treated with theG.lucidum-MRS medium.In addition,we detected phagocytes labeled F4/80 and measured the ratio of CD4+to CD8+,but there was no statistically significant difference between the groups(Figure 6C).

3.7 G.lucidum-Bacillus subtilis fermentation broth regulated the intestinal dysbiosis in ceftriaxonetreated mice

By analyzing the v3-v4 region of the 16S rRNA gene sequence,we assessed the composition of intestinal microbiota in four groups of mice.The values obtained for Shannon’s index indicate that the alpha diversity of intestinal microbiota of mice in the CS group was apparently lower than that in the CON group,and the alpha diversity of mice in the FT group was significantly increased compared with the CS group(Figure 7A).By means of nonmetric multidimensional scaling(NMDS)analysis,we found that after treatment with the fermented broth,the FT and CON groups showed no obvious difference in microbiota diversity,whereas there was a significant difference between the CS and GL groups with CON group(Figure 7B).After application of ceftriaxone sodium,the CS group harbored more abundantEnterococcus(25.48%±0.059%),probably due to the resistance ofEnterococcusspecies to many antimicrobial agents including ceftriaxone.Treatment with theG.lucidum-Bacillus subtilisfermentation broth inhibited the excessive increase ofEnterococcusin mice(0.82%±0.003 6%); the relative abundance ofEnterococcusin the treatment group was similar to that of the CON group(0.20%±0.000 4%).The relative abundance ofLactobacillusincreased in the FT and GL groups(15.03%±0.029% and 12.50%±0.034 8%,respectively)compared with the CS group(8.67%±0.064%).Interestingly,Lachnoclostridiumwas also a prominent genus of bacteria in the gut of FT and GL groups of mice,accounting for 9.50%±0.036 8% and 7.22%±0.03%,respectively,of the total bacterial population(Figure 7C).Furthermore,the linear discriminant analysis effect size(LEfSe)was used to distinguish the microbiota with significant abundance difference(P＜0.05)in each group.As shown in Figure 7D,the generaAkkermansia,BlautiaandRoseburiaare significantly more abundant in the GL group than in the CS group.These genera,especiallyAkkermansia,contributed significantly to the improvement in abundance of the phylum Verrucomicrobia in the mice.On the other hand,mice treated with theG.lucidum-Bacillus subtilisfermentation broth showed an increase in the relative abundance of gutLactobacillusspp.,which may have contributed significantly to the improvement of the abundance ofBacteroidetesin the mice.In addition,in both FT and GL groups,a significantly more taxa in the Ruminococcaceae family were found,which suggests a regulatory effect ofG.lucidumon the gut microbiota of mice.

4 Discussion

In the clinic,many antibiotics,such as ceftriaxone sodium,have been used for various purposes,from growth regulation to disease treatment[21,22].However,because host microorganisms can become resistant to antibiotics,it is necessary to avoid overuse of antibiotics[23].It has also been reported that antibiotics can significantly reduce the density of the intestinal microbiota and alter the host intestinal microbiota composition in the long term[24,25].The resulting reduction in signals from the intestinal mucosa and surrounding organs leads to impaired immune system function,which in turn causes the host to develop multiple organ diseases,such as allergies,infections and inflammation[26,27].Probiotics have a beneficial effect on human health by colonizing the human body,changing the microbiota composition of the host,and generating active substances that balance the effects of pathogenic microorganisms[28,29].Therefore,probiotics are often used to restore the natural microbiota after antibiotic treatment[30,31].In this study,we first established an antibiotic-induced model of mouse intestinal barrier impairment,and then administered to the miceG.lucidummedium andG.lucidum-probiotic fermentation medium to investigate the effect of the intestinal mucosal barrier repair and inflammation relief.

G.lucidumhas many outstanding health benefits and contains more than 400 bioactive compounds including triterpenoids,polysaccharides,nucleotides,sterols,steroids,fatty acids and proteins and peptides[32,33].It has been shown to have effects against tumors[34,35],microbial infection[36],arterial atherosclerosis[37],inflammation,hypolipidemia[38]and diabetes,and is an antioxidant and free radical scavenger with anti-aging,anti-fungal[39]and antiviral effects.Among the most important pharmacologically active compounds are triterpenoids and polysaccharides[40].The polysaccharides ofG.lucidummainly consist of high molecular weight heteropolymers; the main component is glucose,but also present are xylose,mannose,galactose and fructose[41],which have a variety of biological effects such as antiinflammatory,antibacterial,anti-oxidant and anti-tumor[42]effects.Research has shown that polysaccharides extracted and purified fromG.lucidumpolysaccharides containβ-1,3-glucan orα-1,4-linked polymannose,which can significantly inhibit the growth of a variety of pathogenic bacteria(Bacillus cereus,

Bacillus subtilis,Escherichia coli,Aspergillus nigerandRhizopus nigricans)[43].G.lucidumacid(gas),the biologically active ingredient ofG.lucidum,has anti-inflammatory[44],anti-tumor development[45],and causing a decreased concentration of blood lipids.Our HPLC results showed thatG.lucidummedium andG.lucidum-probiotic medium have different triterpenoids.In addition,we found that the pH value of theG.lucidum-probiotic after fermentation is significantly reduced.We speculate thatBacillus subtilisconvertsG.lucidumtriterpenoids to organic acids,thus enhancing the anti-inflammatory effect ofG.lucidum.

Subsequently,we explored the effects ofG.lucidumandG.lucidum-probiotics on antibiotic-induced intestinal barrier imbalance in mice.The results showed that both theG.lucidum-probiotic fermentation and theG.lucidumbroth not only significantly increased the level of anti-inflammatory factor IL-10 and reduced the levels of pro-inflammatory factors TNF-αand IL-6,but also relieved the intestinal barrier damage caused by the antibiotics.In addition,we found that theG.lucidum-probiotic fermentation has better anti-inflammatory and intestinal barrier repair effects thanG.lucidumalone.To explore the specific anti-inflammatory mechanism ofG.lucidum-probiotic fermentation,we used flow cytometry to compare the immune function of antibioticinduced mice before and after the application ofG.lucidumprobiotic fermentation broth.The results showed thatG.lucidum-probiotic medium exerts its anti-inflammatory effect by improving over-regulation of T cells.Interestingly,the application ofG.lucidumfermentation liquid alone could not significantly improve over-regulation of T cells.Therefore,co-fermentation ofG.lucidumand probiotics can significantly enhance the anti-inflammatory effect and repair damage to the intestinal barrier.

After antibiotic administration,the intestinal microbiota of mice was disturbed.The species richness of gut microbiota in mice decreased; the relative abundance ofLactobacillusdecreased,whereas the relative abundance ofEnterococcusincreased.In contrast,the intestinal microbiota characteristics of mice treated with eitherG.lucidumorG.lucidumprobiotic fermentation medium were more similar to the normal microbiome of mice.The intestinal microbiota was characterized byLactobacillusas the dominant bacteria and rarelyEnterococcus.This suggests thatG.lucidumandG.lucidum-probiotic can correct the intestinal microbiota imbalance,which helps maintain a steady state of the microecology within the host organism.Of course,Lactobacillusis one of the most abundant microorganisms in the human gastrointestinal tract and is related to intestinal health.Therefore,Lactobacillusspp.have been widely used as probiotics to ferment foods and make supplements,with the aim of preventing and treating diseases in humans[46,47].A large accumulation ofEnterococcusis found in patients treated with antibiotics[48],and our results are consistent with this finding.We also found,interestingly,that the relative abundance ofBlautiain the intestinal microbiota of mice increased after treatment withG.lucidum.It has been reported thatBlautiahas beneficial effects,such as reducing inflammation and rebalancing the intestinal microbiota[49],which is also evidence of the anti-inflammatory effects ofG.lucidumtreatment.

In conclusion,we found thatG.lucidumcan relieve the intestinal microbiota disturbance and inflammatory response caused by antibiotics in a mice model.At the same time,we also found that the joint fermentation ofG.lucidumwith a strain of probiotics can significantly enhance the anti-inflammatory effect ofG.lucidumand promote the rebalancing of the intestinal microbiota.This suggests that we can enhance the metabolism of Chinese medicinal materials through the co-fermentation of probiotics and Chinese medicinal materials,which is conducive to the development of medicinal effects.

Acknowledgements

We thank for the funding support from the National Natural Science Foundation of China(No.31900920),the Nutrition and Care of Maternal & Child Research Fund Project of Guangzhou Biostime Institute of Nutrition & Care(No.2019BINCMCF02)and the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences,China.

Competing Interests

The authors declare no conflict of interest.