Berberine Modulates Lupus Syndrome via the Regulation of Gut Microbiota in MRL/Lpr Mice

Background Intestinal ora disorder and immune abnormalities have been reported in systemic lupus erythematosus (SLE) patients. Few researches indicated the intestinal status in Chinese SLE patients. Berberine (BBR) showed signicant effects on regulating the intestinal ora, repairing gut barriers and regulating immune cells. This study mainly explored intestinal ora and metabolites in local Chinese SLE patients and the inuence of BBR to MRL/Lpr mice. Methods


Introduction
Systemic Lupus Erythematosus (SLE) is a prototypic autoimmune disease de ned by autoantibody formation, histiocytic in ltration and terminal organ damage. Lupus nephritis (LN) is the most severe organ manifestations and the leading cause of lupus mortality [1,2]. The hyperactivity of autoimmune T and B cells leads to immunoreactions with autoantibodies generation and systemic in ammatory response. Glomerular deposition of autoimmune complexes is the most pathological change in SLE. Systemic in ammatory activation results in acute or chronic systemic in ammatory symptoms, such as gastrointestinal reactions. Thus, down-regulation of activated T and B cells is a legitimate way for SLE treatment [3][4][5].
The thriving development of microbiota research have shown the signi cantly abnormal micro ora structure in SLE patients, such as the increased proportion of Bacteroides and lower Firmicutes/Bacteroidetes (F/B) ratio [6][7][8]. In the study including 61 SLE patients, the micro ora was signi cantly correlated with SLE disease activity index (SLEDAI) [8]. High levels of short-chain-fatty acids (SCFAs) from fecal samples of 21 patients were observed, especially the acetate and propionate [9]. Lactobacillus treatment targeting intestinal ora of MRL/ Lpr mice signi cantly alleviated SLE disease manifestations. The "leaky" gut situation was eased by the increased expression of barrier-tight-function protein [10]. These researches suggested the potential role of gut microbiota in lupus pathogenesis. Meanwhile, modulating gut microbiomes can be a potential therapeutic method to SLE.
Berberine (BBR) is an important natural isoquinoline alkaloid extracted from coptis chinensis and cypress needles [11]. BBR exhibits its surprising therapeutic effects in the regulation of immunoregulation. Meanwhile, intestinal ora plays an essential role in the metabolism of BBR. In the treatment of obese rats [12], BBR showed the ability of signi cantly enriching the SCFA-producing bacteria which is represented by Bacteroides. When used to relieve the symptoms of colitis [13,14], the metabolite of BBR increased the abundance of Bacteroides and regulated intestinal epithelial barrier dysfunction by enhancing the tight junction proteins, ZO-1 and occludin. BBR also increased the proportion of Foxp3 + Treg cells in the spleen and mesenteric lymph nodes (MLN). These results demonstrated that BBR ameliorated diseases via modulating intestinal ora and its metabolites, protecting colonic integrity, and regulating immune cells. Given the existing studies, BBR may be a promising agent worthy to be explored.
In the current study, for the rst time, we analyzed the intestinal ora and metabolites of Chinese SLE patients. Meanwhile, we creatively apply BBR to SLE treatment, monitoring the effect of ameliorating intestinal dysbacteriosis to multiple organ damages and over-activated immune system. Our ndings provide new insights into the treatment of autoimmune diseases.  [15]. Exclusion criteria were listed as follows: 1) Pregnancy or breast-feeding; 2) Recent or current medical disorder (cardiac, respiratory, gastrointestinal, neurological, endocrine, malignancy, etc); 3) History of probiotics within 2 weeks or antibiotics within 3 months before admission; 4) If on immunosuppressant, the dose must be stable for 4 weeks before the sample collection. The gender and age-matched healthy controls (N) were recruited from the Health Examination Centre of A liated Hospital of Nantong University. The inclusion of human participants and supporting documentation were approved by the A liated Hospital of Nantong University Ethics Committee (2017-K002). All fecal specimens of participants were collected in a sealed fecal storage box after defecation and stored at -80°C after collection.

Experimental animal and treatments
Fourty six-week-old female MRL/Lpr mice and ICR mice were bought from Shanghai Sushang Biological Technology Co., LTD. The mice were in the SPF environment of animal research center of Nantong University and were housed three to four per cage at 23-24 ℃ with a 12-h/12-h light/dark cycle with free access to food and water. This animal study was proved by the Institutional Animal Care and Use Committee of Nantong University (S20200313-014).
These mice were divided into ve groups (8 mice/group): control ICR mice (N) and MRL/Lpr mice (S) were treated with saline. The dose of BBR in person is 0.3g-0.9g/day. According to Meeh-Rubner equation After the appearance of lupus nephritis (Fig. 3B), mice were given saline or berberine orally. After 6 weeks , treatment, mice were sacri ced. Serum, faeces and organs samples were collected. The kidneys and colons were formalin-xed and para n-embedded and sliced at 4 µm thickness for further staining. Data analyses were performed by the Sangon platform. The detection of intestinal metabolites, shortchain fatty acids, was carried out by Wuhan Huada Medical Laboratory Co. LTD (supplementary Fig. 1). Thermo Trace1300-Thermo TSQ9000 tandem mass spectrometry and SIM mode were used for forward detection. Trace nder (Thermo Fisher Scienti c, Waltham, MA, USA) was used for data processing. After calculation, the absolute content of target compounds in samples was obtained. The datasets used and analysed during the study are available from the corresponding author.
2.4 Extraction of total DNA of fecal sample and quantitative real-time Polymerase Chain Reaction (qRT-PCR).
The QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Germany) was used to obtain the total DNA from the sample of faeces. The DNA sample was diluted with RNase free water (Beyotime, China) into 5ng/ul and performed the real-time quantitative PCR. According to the bacterial colony 16S rRNA V3 sequence, the speci c primers of total bacteria, Bacteroides and Firmicutes were designed and synthesized by Biomics Biotechnology, China. Sequences and reaction systems were showed in supplementary Tables 1 and 2.
The procedure was operated in the Roche cobas z 480.

Histo-morphological Staining
The colon and kidney sections were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS) and Masson's trichrome respectively (Solarbio, Beijing). HE staining monitored the histological change, PAS staining evaluated the in ammatory cell in ltration and Masson's trichrome staining determined the degree of brosis. Pathological histology was observed by a biological microscope.

Flow cytometry
Lymphocytes were isolated from the tissue homogenates of spleen and mesenteric lymph nodes (MLN). The following primary antibodies were used in the process: mouse BV510-CD3 (Cat. 100233, BioLegend),

Statistical Analysis
Quantitative data were expressed as mean ± S.E.M. Differences determined by two-tailed t-test were used for two-group comparisons and One-way ANOVA was used for multiple comparisons. P-values < 0.05 indicated the experimental results are reliable.

3.1
The intestinal ora and metabolites in uenced the disease activity of Chinese SLE patients.
In our study, 16S high-throughput sequencing were conducted in faeces from 40 SLE patients and 26 normal people. There was no signi cant difference between community richness (Fig. 1A) and diversity (Fig. 1B) in two groups. Gut microbiota in two groups showed the similar composition (Fig. 1C). It is noteworthy that SLE patients showed higher abundance of Bacteroidetes and lower abundance of Firmcutes (Fig. 1C). Similar ora composition showed in the MRL/Lpr mice which indicated the MRL/Lpr mice could be used as dysbacteriosis model of SLE (Fig. 5C).
Then, Stool samples from 64 normal people and 64 SLE patients were collected to validated the above conclusion. The results of qRT-PCR showed higher Firmcutes/Bacteroidetes (F/B) ratio in SLE patients (Fig. 1D). The F/B ratio was negatively correlated with SLEDAI score (Fig. 1E). These results substantiated the dysbacteriosis in SLE patients and its relationship in disease development.
Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) technique was applied to identify functional categories (Fig. 2). And the related functions such as immune system diseases, membrane transport, amino acid metabolism were quite different between two groups. Gas chromatography technology was applied to analysis the metabolism (Table 1). Almost all the tested SCFAs found statistically signi cant results in SLE and LN patients, especially the propanoic acid and butyric acid. We could conclude that dysbacteriosis and abnormal metabolism can in uence Chinese lupus pathogenesis. Table 1 Analysis of fecal short-chain fatty acids in healthy controls and SLE patients with different disease activity. The differences in speci c short-chain fatty acids between healthy controls (N), SLE patients (S) and SLE patients with lupus nephritis (LN) were assessed by gas chromatography-mass spectrometry (GC-MS In this study, berberine was used to treat the animal model of SLE-MRL/Lpr mice. At the end of procedure, body weight was not statistically different between BBR treatment and saline treatment (Fig. 3C). The decrease of urine protein suggesting the relieving effect of berberine on lupus nephritis (Fig. 3D). The concentration of dsDNA and ANA antibodies was increased in the serum of BBR treated MRL/Lpr mice, while the complement C3 and C4 was decreased (Fig. 3E-H). Next, we assessed renal histopathological changes (Fig. 4A). The HE, PAS and Masson staining results demonstrated serious mesentery proliferation, in ammatory cell in ltration and renal interstitial in MRL/Lpr mice. We found that BBR gradually alleviated kidney pathological changes in MRL/lpr mice in a dose-depended manner. The immune complex-induced vascular in ammation are key mechanisms in the development of LN [17].
The classical generation of C3 is originated by C1q directly binding to complement-xing antibodies-IgM and IgG. Glomerular deposition of C3, IgG and IgM were signi cantly high in MRL/Lpr mice (Fig. 4B). The deposition of complement C3 and immunoglobulin IgM and IgG in all BBR treatment groups were signi cantly reduced compared with control MRL/Lpr mice. These results indicated that BBR treatment relieved autoantibody secretion, alleviated nephritis conditions and ameliorated the renal pathological damage of MRL/Lpr mice.

Berberine regulated intestinal ora and metabolites in MRL/Lpr mice.
It has been reported that berberine regulated intestinal ora as an anti-in ammatory agent, especially increasing the proportion of Bacteroides [18,19]. MRL/Lpr mice showed increasing gut microbiota richness and diversity, while BBR signi cantly abrogated the differences in MRL/lpr mice (Fig. 5A, B). At the phylum level (Fig. 5C), Bacteroidetes and Verrucomicrobia was increased gradually in MRL/lpr mice treated with BBR at all dose. Firmicutes was decreased distinctly in the MRL/lpr mice treated with BBR at medium and high dose, where Bacteroidetes became the dominant bacteria. As the result of ora adjustment, the expression of major SCFAs in fecal sample surprisingly increased, especially the butyric acid and n-valeric acid ( Table 2).   Mechanical barrier refers to the intact intestinal mucosal epithelial cells and the tight connections between the epithelial cells [20,21]. The main manifestations of MRL/Lpr mice were destruction of epithelial structure and goblet cells. The intervention of berberine completed the intestinal tissue structure tends, rearranged goblet cells and reduced the intestinal in ammation, especially in MRL/Lpr mice treated with high BBR dose (Fig. 6A).
Western blot was used to analysis the protein ZO-1 and occludin (Fig. 6B, C). The MRL/Lpr mice showed signi cant de ciency of both proteins, while the protein expression level of ZO-1 increased signi cantly in group M and H and the expression of occludin increased in all BBR-treated mice. Immuno uorescence staining showed that MRL/Lpr mice were also de cient in related proteins, while in all BBR-treated groups, ZO-1 and occludin were signi cantly enhanced (Fig, 6D). The application of berberine enhance the intestinal barrier function.

Berberine regulated immune function in MRL/Lpr mice.
Immune manifestation of SLE is the most direct embodiment of the abnormal activation of the immune system. The abnormal activation of T and B cells directly leads to the harmful changes in immune functions [22][23][24][25]. As shown in Fig. 7A, B, the decreased spleen index and lymph nodes index directly showed the amelioration of immune activation in the BBR-treated group. Smaller spleen size was quite obviously in group M and H (Fig. 7C).
Lymphocytes from MLN and spleen were obtained and detected by ow cytometry. Statistical results indicated that Treg and Tfr cells were increased in the MRL/Lpr spleen after the berberine treatment ( Fig. 7D, F). Meanwhile, the proportion of Treg cells in MLN was enhanced (Fig. 7E, G). However, no signi cant results were found for B cells and NK cells (supplementary Fig. 2, 3).

Discussion
Previous studies have reported interactions between the gut microbiota and alterant homeostatic balance in SLE patients. Further researches revealed the correlation between the abnormal abundance of the Firmcutes and Bacteroidetes and disease activity in SLE [8,26]. Gut microbiota are associated with the pathogenesis of disease through intestinal metabolites-SCFAs [27,28], While few reports mentioned the abnormal gut metabolites in SLE patients [9]. Our investigation, for the rst time, illustrated the relationships between gut microbiota, intestinal metabolites and disease activity in Chinese SLE patients.
In MRL/Lpr mice, berberine signi cantly eased splenomegaly and increased Treg, Tfr in spleen and MLN. Abnormal autoantibodies in the circulation and kidney damage changed, too. Metabolism of berberine is con ned in the gut lumen [29]. Increasing evidences showed that intestinal microbiota is the target of berberine [30]. Like in colitis mice, BBR increased the abundance of Bacteroidetes and ameliorated intestinal epithelial barrier function via enhancing the expressions of tight junction proteins (ZO-1 and occludin) in MRL/Lpr mice. As a member of butyrate-producing communities [31], increasing of Bacteroidetes leads to the production of butyric acids. Butyrate showed the ability of enhancing the intestinal barrier [14,32,33] and facilitating the generation of Treg cells [34,35]. Therefore, the repaired "leaky" gut and a shift of immune balance could explain how berberine remitted lupus disease in MRL/Lpr mice.
The gastrointestinal tract is the largest immune organ which leading the regulation of immune homeostasis. Intestinal epithelial barrier showed strong interactions with the gut microbiome and immune system [36]. Recent studies indicated composition of the gut microbiome modulated metabolites, which affect the gut barrier. It is noteworthy that intestinal barrier disruption can be related to increased susceptibility to immune diseases [20,37]. Current studies con rmed the dysregulation of Treg cells prevent autoimmune diseases and maintain a stable immune state [39].Treg cells were considered to be inhibitors of lupus disease, which resulted in delayed disease progression and reduced mortality in lupus prone mice [40]. Increasing Treg cells proportion was observed with Berberine treatment [41,42]. Butyrate promotes differentiation of Foxp3 + Treg cells in vivo [34]. Tfr cells compete intensively with Tfh on germinal center B cells, promoting the production of high-a nity antibodies and limiting the overall expansion of antigen-speci c B cell clones [43]. In mice model, the loss of Tfr cells leads to the proliferation of Tfh and GCB cells, as well as the production of antibodies [44,45]. Those fact may explain the increased Tfr cells in berberine treated mice.
Together with the change of immune cells, BBR treatment do improve lupus nephropathy in MPL/lpr mice. The level of anti-dsDNA antibodies can re ect disease activity, while antinuclear antibodies (ANAs) mediated tissue deposition of immune complexes [2,46]. Serum C3 and C4 were correlated with disease activity to some degree [47]. Low C3 was associated with renal involvement and poor renal prognosis [48]. Our results showed that BBR was able to ease nephropathy in MRL/Lpr mice as reducing level of urine protein, serum immunological markers and improving kidney function. In addition, BBR was able to down-regulate the urine protein, which indicated increase proximal tubular reabsorption and glomerular ltration rate. Furthermore, BBR show the ability of inhibiting complement cascade via preventing the consumption of serum C3 and C4. The effect of BBR on lupus nephritis was con rm by histopathological study and immuno uorescence. BBR was able to improve the damaged kidney to the relatively normal glomerular structure and inhibit the levels of local C3, IgG and IgM.
Our study highlights current status of intestinal dysbacteriosis in Chinese patients with SLE and differences in intestinal metabolites among patients with different disease states. The regulation of intestinal ora and the repairment of gut barrier by intestinal metabolites in BBR treated mice seemed to be the factor that directed the immune responses and disease outcomes (Fig. 8).

Conclusions
In conclusion, gut microbiome regulated the disease development in Chinese SLE patients. Also, BBR treatment modulates the lupus syndrome via the regulation of gut microbiota. The application of berberine is a relatively safe and convenient way. However, it is di cult to promote this method due to the complexity of individual ora and diverse micro ora in different disease activities. Therefore, further investigations will focus on the effects of berberine and its metabolites on intestinal function and systemic immunity.

Consent for publication
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Availability of data and materials
The data underlying this article are available in the article and supplementary material.

Competing interests
There were no competing interests between the authors.     Gut microbiota and metabolites were abnormal in Chinese SLE patients. The rapeutic effects of berberine in MRL/Lpr mice. Chinese SLE patients showed the increased Bacteroidetes and abnormal SCFAs in gut.