Potential ecacy of gut microbiome dysbiosis on alleviation the progress of osteoarthritis in mice

Objective To evaluate the relationship between the gut microbiota dysbiosis and progression of osteoarthritis (OA) Design We used a mice OA model (8 weeks) of destabilization of medial meniscus and the gut microbiome dysbiosis induced by antibiotic treatment(ABT) with ampicillin and neomycin for 8 weeks. The severity of OA was evaluated by micro-CT, X-ray and histology of osteoarthritis Research International (OARSI) score. Microbiome analysis by 16 s DNA qPCR was performed on the feces samples and the serum IL-6, TNF-α, osteocalcin, estrogen, calcium and magnesium ion were measured in enzyme-linked immunosorbent assay(ELISA).


Introduction
Osteoarthritis (OA) is the most common musculoskeletal diseased and most notable increase of 35% in total burden and 4% in age-standardized disability-adjusted life-years rates [1,2]and knee OA accounts for approximately 85% of the burden of osteoarthritis worldwide. For knee OA, strong evidence indicates a variety of moderate to strong risk factors, including menopause ,obesity and previous knee injury [3,4]and associated with metabolic disease, for example high levels of obesity [5].
Gut ora is also a critical determinant of metabolic disease and can be modulated by the combined effects of environmental stimuli and genetic factors [6]and also combine with various conditions including obesity, diabetes, irritable bowel syndrome, in ammatory bowel disease, depression, and cardiovascular disease [7]. The gastrointestinal tract microbiota pro le in uence nutraceutical e cacy in a non-blinded randomized clinical trial [8].The current literature on gut ora and OA suggest that gut microbiome-OA connection sets the stage for discovery of potentially OA of obesity involving strategic manipulation of speci c microbial species inhabiting the intestinal space [9]. Current gut ora mechanisms for improved mineral absorption and skeletal health include alterations in gut microbiota composition, production of short-chain fatty acids, altered intestinal pH, biomarker modi cation, immune system regulation, vitamin D and vitamin K [10,11].
The effects of gut microbial dysbiosis on musculoskeletal tissue during and after antibiotic treatment in human and animal had attracted the attention of researchers [12,13]. Several researchers have explored the effect associated with gut microbiota dysbiosis and human life system. Dysbiosis in the composition and abundance of gut microbiota can affect microbiota-gut-brain axis and gut-liver axis [14,15]. There are two methods of gut microbial dysbiosis, such as antibiotic treatment and germ-free feeding. For the germ-free mice model of destabilized medial meniscus (DMM), these results suggest factors related to the gut microbiota promote the development of OA after joint injury in subchondral bone [16]. Otherwise, Previous studies with intestinal microbiota have also shown that osteoarthritis is characterized by an expansion and/or decrease of bacterial groups in obesity [6]. What's more, imbalances in the gut microbiota, the bacteria that inhabit the intestines, are central to the pathogenesis of obesity [17]. Recent evidence suggests that antibiotic treatment may cause profound alterations of gut epithelium, immune cells, intestinal neural system and long-lasting effects for the host [18]and it might explain why and how antibiotic-caused microbiota changes can impact OA pathogenesis in obesity [19,20]. The two major in ammatory phenotypes of macrophages are controlled by gut ora through differential consumption of glucose, glutamine, and oxygen.M1 phenotype is triggered by polarization signal from bacterial lipopolysaccharide and Th1 proin ammatory cytokines such as interferon-γ, TNF-α, and IL-1β, or both, whereas M2 phenotype is triggered by Th2 cytokines such as interleukin-4 and interleukin-13 as well as anti-in ammatory cytokines, IL-6 [21]. But for health people, the in uence of antibiotics-induced gut microbiota dysbiosis on OA and how the effects are modulated by diet are poorly understood [22].
We hypothesized that antibiotic depletion gut ora which could reduce a source of microbial products that mean suppress innate immune response to restrain chronic low-grade proin ammatory state and postpone the development of OA. We explore the associations between gut ora, estrogen, systemic in ammation pro les and joint damage in antibiotics-induced dysbiosis mice with the purpose of better understand the development of OA.

Experimental animals and study design
Mice used in this study were 8 weeks and purchased from Department of Laboratory Animal Science of Peking University Health Science Center. The experiment and protocol were approved by The Peking University Third Hospital Committee on Ethics in the Care and Use of Laboratory Animals(No. LA2019209). Total of thirty-six C57BL/6N mice (Male and female matching) were housed in a standard animal facility under controlled temperature (22℃) and photoperiod (12 h light and 12 h dark) and had free access to fresh water and food. The mice were rst treated with knee surgery, and interventions measure were given one week after the surgery. Animals were allocated to antibiotics-induced group (ABT) (ampicillin(1.0 g/L) and neomycin (0.5 g/L) or the standard control group (drinking fresh water) lasted for 8 weeks [23].

Induction Of Destabilization Of The Medial Meniscus (DMM) Surgery
Methods and results description follow Animal Research Reporting in Vivo Experiments guidelines and DMM was induced by transecting the medial meniscotibial ligament in the right knee as described by Dr.
Glassion [24,25]. We performed the DMM procedure on one knee in all mice and used the unoperated left leg as a control instead of performing sham surgery in a separate group of animals which in pervious studies [16]. Mice subjected to DMM/sham were assessed for weight, fat percent in 16 weeks after surgery. Dissected knees were xed, micro-CT scanned and then histology. Blood and stool samples were collected.
Blood Collection And Serum IL-6,TNF-α,OCN And Estrogen Measurements Blood was collected in coagulation-promoting tubes and centrifuged at 3000 rpm for 15 min and plasma were collected and stored in -80℃. Plasma IL-6,TNF-α,OCN and estrogen(Meimian Biotechnology ,Yancheng, Jiangsu, China) were performed using commercial enzyme-linked immunosorbent assay (ELISA) kits. the level of Calcium ion and magnesium ion were quanti cation by Methyl thymol blue microplate method(Shanghai yuanye Bio-Technology Co., Ltd)

Radiological Anslysis
Dissected knees were xed overnight(4% paraformaldehyde). The specimens were scanned in a sample holder with PBS and scanned using a soft X-ray apparatus with a small-animal high-resolution collimator (Faxitron® LX-60 Cabinet radiography system, US) and µ-CT(Inveon, Siemens, Erlangen, Germany) at a spatial resolution of 55 kVp, 145 mA, 300 ms integration time, and a 6-mm isotropic voxel edge. A region of interest contain the medial tibial plateau which de ned as the region of the tibia superior to the growth plate, posterior to the insertion of the anterior cruciate ligament (ACL), and medial to the midline of the intercondylar notch. We analysis the subchondral bone sclerosis and remodeling in this region with a direct-model morphometric measure of bone volume (BV) over total volume (TV), trabecular thickness(Tb.Th), trabecular number(Tb.N), and trabecular spacing(Tb.Sp) in the medial tibial subchondral trabecular bone which has been describe in pervious studies [26]. Peripheral osteophytes were not analysis in µ-CT.

Histology Assessment
Fixed knees were decalci ed in 10% EDTA for 14 days and embedded in para n and frontal sections(5 µm). The Safranin-O/Fast Green staining and HE were subjected and the images were taken a stereomicroscope (SMZ745T, Nikon, Tokyo, Japan). For OA grading, we used the OARSI staining histopathology score for evaluating the OA severity [27]. Osteophyte size and osteophyte maturity were scored on coded digital images of the same location of the anterior-medial tibia in each animal [28]. All histological scores were found by an evaluator (ZYG) blinded to the group assignment and we reported the OARSI score either medial femoral condyle, medial tibial plateau, lateral femoral condyle using ImageJ and the results were reported as average.

Microbiome Analysis At
At age 16 weeks, the control and ABT mice were killed and caeca sample were collected and frozen at -80℃.Fecal sample were isolated by the Qiagen QIAamp DNA Mini Kit, as directed by the manufacturer (Qiagen, Hilden, Germany) and total concentration of DNA were measure by NanoDrop (ThermoFisher, Waltham,USA). The total bacterial load and speci city bacterial load were tested by quantitative PCR(qPCR) in previous studies [29][30][31]. The universal primer and speci city bacterial perimer include αproteobacteria, γ-proteobacteria, Bacteroidetes, Firmicutes, actinobacteria (Aokedingsheng Technologies,Beijing,China) were upload in supplement table1. The qPCR protocol was completed on ABT StepOne (Applied Biosystems) and qPCRs contained 2Fast SYBR Green Master Mix (Applied Biosystems), 0.25 mmol/L primers, and 150 mg/mL DNA template. Samples underwent an initial denaturing step at 95℃ for 5minutes, followed by 30 cycles of 95℃ for 30 seconds, 60℃ for 40 seconds, and 72℃ for 30 seconds, and ending with a nal elongation step of 72℃ for 5 minutes.

Statistical analysis
All measurements are presented as the mean ± standard deviation (SD) and a P-value of ≤ 0.05 was considered statistically signi cant. The data were analyzed using GraphPad Prism 8.02(La Jolla California USA) and one-way ANOVA followed by Tukey's multiple.
3. Result 1.Flora analysis following dysbiosis induced by ABT treatment in mice Antibiotics and control-treatment was administered via drinking water to adult sex-matched C57BL/6T mice with DMM surgery (Fig. 1A).In order to dysbiosis the intestinal microbiota, 8-week-old C57BL/7 mice were treated by broad-spectrum antibiotics though 16 s DNA qPCR analysis (Fig. 1B-F).ABT decreased total load of colonic bacterial in female and male(P < 0.001 for both analysis, Fig. 1B)mice than control group. These are sign cant difference in control-female ,ABT-female, control-male and ABT-female in subgroup analysis (Fig. 1C).Female antibiotic and control-treated mice demonstrated signi cant increases in α-proteobacteria (P < 0.05 for both analysis, Fig. 1F) and bacteroidetes(P < 0.01 for both analysis, Fig. 1F) and signi cant decrease γ-proteobacteria (P < 0.01, Fig. 1G).Male antibiotic versus control-treated mice showed a signi cant decrease in Firmicutes(P < 0.01, Fig. 1G).
2.Antibiotic-induced dysbiosis causes decrease subchondral bone sclerosis, osteophyte formation and improve cartilage damage via gut barrier dysfunction To understand the effect of ABT on the subchondral bone sclerosis, the subchondral bone sclerosis also has been analysis after DMM surgery by micro-CT scan and analysis. The Fig. 2A-B obtained from the preliminary analysis of micro-CT of the joint in DMM. The result show that ABT inhibit subchondral bone sclerosis in BV/TV in both female and male mice (P < 0.05 for both subgroup analysis, Fig. 2E) and also found that ABT treatment have signi cant difference between male and female mice(P < 0.01, Fig. 2E).Micro-CT demonstrated that female ABT signi cant increased the subchondral trabecular thickness (P < 0.01 for both subgroup analysis, Fig. 2F ) and signi cant decreased trabecular number (P < 0.01 for both subgroup analysis, Fig. 2C)and trabecular separation (P < 0.01 for both subgroup analysis, Fig. 2D). To the contrary, male ABT signi cant decreased trabecular separation(P < 0.01 for both subgroup analysis, Fig. 2D). Figure 3 shows osteophyte formation in knee joint via a X-ray apparatus and HE staining. Obvious osteophytes, uneven joint surfaces, and narrowed joint spaces has been observed in radiological images and osteophyte formation were indicated by black bar in HE staining images (Fig. 3A-3B).ABT treatment has decreased the osteophyte score in female mice after DMM surgery(P < 0.05, Fig. 3C) and also declined osteophyte maturity score in both female(P < 0.01, Fig. 3D) and male(P < 0.05, Fig. 3D)mice. ABT treatment have signi cant difference between male and female mice in osteophyte score. (P < 0.01, Fig. 3E) An overview of cartilage damage in joint via Safranin-O/Fast Green staining and the OARSI score has used to analysis the medial femoral condyle, medial tibial plateau, lateral femoral condyle in Fig. 4. ABT treatment has decreased the OARSI score in medial tibial plateau (P < 0.01 for both subgroup analysis, Fig. 4C) and medial femoral condyle (P < 0.01 for both subgroup analysis, Fig. 4C) in both male and female mice. ABT treatment also improve in lateral femoral condyl (P < 0.01 for both subgroup analysis,

ABT decreased the in ammation and osteoblasts
In order to determine whether in ammation play a role in injury-induced OA development, circulating TNFα (P < 0.01, Fig. 5B)and IL-6 (P < 0.01, Fig. 5C) levels were decreased signi cant in antibiotic-treatment verse control-treated subgroup in both male and female mice. As a marker of bone formation, we measured decrease levels in serum osteocalcin (OCN) in antibiotic mice compared with control-treated mice in both male and female mice (P < 0.01, Fig. 5A). To investigate the gut ora mechanisms for mineral absorption in ABT mice, we measured serum calcium (Ca+) and magnesium (Mg2+) level. Serum calcium has been decreased signi cant in ABT mice with control-treated mice(P < 0.01, Fig. 5D) and we also found the serum magnesium has been increased signi cant in ABT female mice compared with control female mice and no signi cant differences were seen in male mice(P < 0.01, Fig. 5E). To understanding whether the gender can affect the development of OA, we also found that antibiotic also has increased the level of serum estrogen compared with control group in female mice (P < 0.01, Fig. 5F). Pearson's correlation analyses demonstrated that subchondral bone BV/TV(r = 0.6115, P = 0.0000, Fig. 5G) and OARSI score in medial tibial plateau(r = 0.5861, P = 0.0002, Fig. 5H) are positively correlated with the Firmicutes : Bacteroidetes ratio.

Discussion
In this study, we evaluated the effect of antibiotic on the intestinal microbial composition and the relationship between the gut microbiota dysbiosis and progression of osteoarthritis (OA). The concentration of serum OCN, TNF-α and IL-6, calcium was signi cantly decrease with a marked change in gut Flora in 8 weeks of oval antibiotic administration in OA. In contrast, the concentration of serum magnesium and estrogen were increased in antibiotic induced female mice. The results shown that antibiotic change intestinal bacterial and imply that antibiotic decrease the OA development, which may be caused by restrain its in ammatory reaction Antibiotics alter the composition and functions of the microbiota and produce long-lasting effects for host [18],such as education of the immune system [32,33], colonization resistance [34,35] and allergic and metabolic syndromes [36]. In the intestinal, the gut microbiota catalyzes the conversion of drugs into absorbable, hydroxylation, and reduction [37]. We choose ampicillin and neomycin with less absorption in the intestine as an intervention. Another experiment of interfere with the intestinal ora is observe changes in osteoarthritis using germ-free mice. Ulici et.al found that GF mice will suppress the gut microbiota and decrease of development of OA after joint injury [16].
There are many methods to measure the severity of OA, such as OARSI, the Articular Cartilage Structure score, Safranin-O stains score and radiological test. Histopathology is currently the gold standard for assessing of OA in animal models [38]. This semi-quantitative scoring system proposed in this study was relatively easy and reproducibility to apply for both experienced and novices scores [39]. The micro-CT is another powerful technique utilized to study 3D structures reconstructed. Botter suggest that osteoarthritis in the murine knee using collagenase injections has been show signi cant changes in subchondral bone architecture could be detected and quanti ed in 3D with micro-CT analysis [40]. Soft Xray apparatus also the important method to evaluate the severity of OA [41].
In early stages of OA in humans, elevated bone remodeling, subchondral bone loss and articular cartilage regression was observed, and was considered as a determinant of OA progression [42], subchondral sclerosis may be observed only during more advanced stages of OA [43]. In our studies, we found that antibiotic-induced dysbiosis for 8 weeks contribute more advanced stages of OA. There are three potential mechanisms by which the gut microbiota can in uence bone tissues: 1)regulation of nutrient absorption at the gut epithelium, 2)regulation of the mucosal and systemic in ammation and immune system, 3)translocation of microbial contents across the gut endothelial barrier [44]. The overproduction of cytokines and growth factors is the important mediator in the pathophysiology of OA [45]. In OA pathogenesis particularly in synovial in ammation, activated chondrocytes and synovial broblasts (TNF-α and IL-6) have been identi ed as vital players [46]. Zhao et.al found that both spontaneous and surgically induced OA models indicated that TNF-α led to an accelerated OA-like phenotype [47]. TNF-α control the degeneration of articular cartilage matrix, which makes them prime targets for therapeutic strategies [48]. Latourte et.al found that IL-6 induces chondrocyte catabolism mainly via Stat3 signalling, a pathway activated in cartilage from joint subjected to DMM [49]. Intra-articular injections of IL-6 reproduced OA-like cartilage lesions in mice, and IL-6 knockout (KO) mice develop less severe posttraumatic OA lesions [50]. Lu J, et al investigated that correlation between the amount of intestinal ora and the number of CD4 + T lymphocytes and the levels of TNF-α and IL-6 [51]. In our study, we found that antibiotics reduce the in ammatory response throughout the body after inhibiting the intestinal ora. This also provides new evidence for the intestinal ora-joint axis in osteoarthritis.
The role of the intestinal ora in the absorption of substances is very important, such as the absorption of inorganic and energy substances in the host. Scholz-Ahrens et.al studied that probiotic can increase the level of calcium prevented the decrease in bone metabolism in ovariectomized rats and the main mechanism of this process is increased intestinal absorptive bones and increased bowel microbial mass and reduced turnover of calcium in bones [52]. Whisner et.al studies the prebiotic increases calcium absorption, which may be mediated by the gut microbiota [53]. In our studies, we investigated that antibiotic decrease the total load of gut microbiota and it also mean that interaction mechanisms between intestinal ora and minerals [54]. Antibiotics perturbed gut microbial composition. Community diversity and richness were reduced, and the phyla Firmicutes/Bacteroidetes ratio was decreased by antibiotics [55]. This process leads to impaired gut barrier integrity and release of LPS from intestinal gram-negative bacteria for example Bacteroidetes into the bloodstream [56].Therefore, we found that the decrease of community diversity was increase the subchondral bone sclerosis and cartilage damage.
Gender was also reported speci cally in uence subchondral bone. Men are reported to have a higher prevalence of OA than women before the age of 50 years, but after this age the prevalence is higher in women, which coincides with menopause [57]. The estrogen replacement therapy exhibit a reduced risk of hip and knee OA in postmenopausal women [58]. Son et.al found that up of estrogen abrogates experimental OA via the estrogen-related receptor family of orphan nuclear receptor [59]. A lack of estrogens increases subchondral bone remodeling, and this bone remodeling has been suggested to ultimately induce osteoarthritis changes [60]. In general, acute loss of estrogens increases the levels of reactive oxygen species and activates nuclear factor-κB and pro-in ammatory cytokine production, indicating their predominant anti-in ammatory properties [61] .In our previous studies, we found that de ciency of estrogen can accelerate the development of OA which induced by ovariectomy [62]. What's more, antibiotic-induced dysbiosis also increased the level of estrogen which may paly positive role in OA.
The role of osteocalcin in bone remodeling, speci c expression and regulation in osteoblasts which is important biomarker in osteoporosis and level of reduced Osteocalcin expression were maintained in human osteoarthritic chondrocytes and represented a suitable biomarker of osteoarthritic chondrocyte activation [63]. Wagatsuma et.al found that diversity of gut microbiota affecting serum level of osteocalcin in patient [64]. It is means that the occurrence of osteoporosis causes uneven cartilage stress, which further aggravates joints and cartilage damage which make knee osteoarthritis more serious [57].At the same time, the exacerbation of osteoarthritis reduces the activity of mice and reduces stress stimuli which further aggravated osteoporosis [58]. In our studies, we found that antibiotic treatment of gut ora reduced the level of OCN which may contribute to development of OA and osteoporosis.
In addition, the effect of gut ora is also considered important factors for mice with sexual maturity [65]. However, the osteoarthritis after osteoporosis is also common in the clinical.

Conclusion
In summary, by inducing the intestinal microbiota dysbiosis that lead to improvement of OA, these results can help in leading to strategies for new treatments to prevent the symptoms and long-term sequelae of OA. In addition, this study contributes to our understanding of gut ora-joint axis in aspect of OA and provide the relationship between in ammation response, gender and absorption of substances. Further studies of the differences in the transcriptional response of antibiotic treatment should help elucidate the mechanisms of OA and osteoporosis by ABT underlying the contribution of the development of OA.

Competing interests
Authors have no con ict of interest to disclose.   Antibiotic-induced dysbiosis causes decrease subchondral bone sclerosis via gut barrier dysfunction. A and B, Bone sclerosis induced by DMM was measured by micro-CT at the medial tibia plateau including 3D and coronal axis in female(A)and male(B)mice. bone volume over total volume (BV/TV), trabecular thickness(Tb.Th), trabecular number(Tb.N), and trabecular spacing(Tb.Sp) were analysis from sections obtained from the anterior/medial margin of the tibia from DMM-operated knees at 8 wks(C,D,E,F). n=9 per group. Con,control group. Data are expressed as means ± standard deviation. Antibiotic-induced dysbiosis causes decrease osteophyte formation via gut barrier dysfunction. A and B, anterior subluxation of the tibiae, joint destruction and osteophyte formation were visible in the posterior tibial plateau by lateral X-ray features of the knee joints and HE staining. Osteophyte size(C) and Osteophyte maturity score(D) represent decrease in osteophyte formation and cartilage destruction. Black and red bars indicate osteophyte formation. Data are expressed as means ± standard deviation. n=9 per group. Con,control group Antibiotic-induced dysbiosis causes improve cartilage damage via gut barrier dysfunction. A and B, Cartilage damage induced by DMM was measured by OARSI score at the medial tibia plateau in female(A)and male(B)mice. medial femoral condyle (MFC), medial tibial plateau (MTP), lateral femoral condyle (LFC)were analysis from sections obtained from the anterior/medial margin of the tibia from DMM-operated knees at 8 wks(C,D,E). Red bars indicate osteophyte formation. Data are expressed as means ± standard deviation. n=9 per group. Con,control group