Effects of Lactobacillus salivary on spleen T cells and inflammatory factors in a rat model of type II collagen induced arthritis Running title: Effects exploration of Lactobacillus salivary

doi:10.21203/rs.3.rs-4297536/v1

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Effects of Lactobacillus salivary on spleen T cells and inflammatory factors in a rat model of type II collagen induced arthritis Running title: Effects exploration of Lactobacillus salivary

https://doi.org/10.21203/rs.3.rs-4297536/v1

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To explore the therapeutic effect and role of Lactobacillus salivarius on rheumatoid arthritis. A Collagen induced arthritis (CIA) model was constructed in male Sprague Dawley (SD) rats by adopting bovine type II collagen as immunogen. The modeled rats (24 SD rats) were randomly divided into the CIA model group, salivary lactobacillus group, and methotrexate group, and 8 rats in each group with healthy group. Both the healthy group and CIA group were administered bacterial culture medium; the salivary lactobacillus group administered salivary lactobacillus. The methotrexate group given methotrexate medication, and each group was continuously operated by gavage for 4 weeks. After that, evaluated arthritis index, thickness of the affected foot, pathological changes in the naked joint tissue, and histopathological staining. Moreover, the content of T helper cell 17 (Th17) and regulatory T cells (Treg) in the spleen were also detected, and included the levels of expression of interleukin-1 β (IL-1 β), Interleukin-10 (IL-10), Interleukin-17 (IL-17), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-8 (IL-8), γ Interferon (IFN- γ), and Tumor Necrosis Factor- α (TNF- α) in the serum of each group. Compared with the CIA model group, the arthritis index score, onset foot thickness, spleen Th17 cells, the content of inflammatory factors above were significantly decreased (P < 0.05) in the salivary lactobacillus group and methotrexate group. The level of Th17 cells significantly increased (F-value: 238.918, P < 0.05), and the content of Treg cells significantly increased (CIA group: 9.47 ± 0.30, Salivary Lactobacillus group: 13.04 ± 0.47, Methotrexate group: 22.41 ± 0.33, P = 0.003 < 0.05); while the Th17/Treg cell ratio in both the salivary lactobacillus group and the methotrexate group was decreased (CIA group: 0.62 ± 0.03, salivary lactobacillus group: 0.29 ± 0.11, methotrexate group: 0.15 ± 0.03, P = 0.004 < 0.05). However, there was no significant difference in the content of spleen Th17 cells, Th17/Treg ratio, and total joint pathology score (P = 0.54 > 0.05) between the salivary lactobacillus group and the methotrexate group. Lactobacillus salivarius has immune regulatory and anti-inflammatory effects on CIA model SD rats, and the corresponding mechanism of action is mainly reflected in the regulation of Th17 and Treg cell balance, and inhibiting the expression of inflammatory factors.

Biological sciences/Immunology

Health sciences/Rheumatology

Type II collagen induced arthritis

Lactobacillus salivary

Inflammatory factors

Helper T cell 17

Regulatory T cells

Rheumatoid arthritis (RA) is a common chronic inflammatory disease in clinical practice and a highly disabling systemic autoimmune disease [1–3]. At the onset of the disease, symmetrical joint inflammation and morning stiffness are the main manifestations. Further development can easily lead to osteoporosis, subchondral bone destruction, chronic inflammation of the synovial membrane within the joint, and joint cartilage diseases, which are characterized by significant damage and long duration of symptoms [4–5]. In clinical practice, general treatment emphasizes rest and joint immobilization, while drug therapy mainly includes slow acting anti rheumatic drugs, non-steroidal anti-inflammatory drugs, and traditional Chinese medicine [6]. Although these drugs can alleviate or improve the patient's condition [7], they cannot fully control the development of RA. And long-term use can easily lead to a series of side effects [8–10], such as methotrexate. Exploring new treatment methods has always been a focus of clinical doctors' attention. Multiple studies have reported that the imbalance between Treg cells and Th17 cells plays a crucial role in the occurrence and development of RA [11], and the two types of cells play an antagonistic role in RA [12]. From this perspective, whether the goal of treating RA can be achieved by regulating the balance of immune cells.

As is well known, dysbiosis of the gut microbiota can lead to the occurrence of various diseases. Research has found that the onset of RA is related to infections such as Escherichia coli, Proteus mirabilis, and Mycobacterium tuberculosis [13]. Peptidoglycan components of the cell wall and DNA degraded by intestinal bacteria have been detected in the joints of RA patients. The use of human intestinal bacterial cell walls can induce arthritis in experimental animals [14], and many other components of intestinal bacteria can also stimulate the secretion of rheumatoid factor antibodies [15]. Sequencing technology has found that gut microbiota plays an important role in regulating the immune system of the body [16]. Disturbance of gut microbiota will lead to changes in gut microbiota diversity, an increase in pathogenic bacteria, and a decrease in beneficial microbiota, leading to the occurrence and development of RA [17]. Salivary Lactobacillus, belonging to the Lactobacillus family and genus, is widely present in the intestines of humans and animals. It is an important member of the gut microbiota and plays an important role in the occurrence and development of various diseases. However, there is limited research on its role in RA.

Salivary Lactobacillus is an important member of the gut microbiota, belonging to the Lactobacillus family and genus. It is widely present in the intestines of humans and animals and plays a crucial role in the occurrence and development of various diseases [18–19]. This study established a SD rat model of Collagen induced arthritis (CIA) induced by type II collagen, and observed the effect of Lactobacillus salivarius on immune Th17 cells and Treg cells in CIA model rats, laying an experimental foundation for gut microbiota treatment of RA and providing exploratory basis.

1.1 Materials preparation

1.1.1 Preparation of Salivary Lactobacillus and Methotrexate Suspension

Preparation of Salivary Lactobacillus and Methotrexate Suspension: Salivary Lactobacillus (product number: ATCC11741): purchased from Beijing Beina Chuanglian Biotechnology Research Institute. The liquid preparation method is: under standard anaerobic conditions, live bacteria are counted after 48 hrs of cultivation. Based on the counting data, the bacterial solution is diluted with sterile physiological saline to produce a liquid with a concentration of 10¹⁰ CFU/mL, which is stored for future use.

Methotrexate suspension: The manufacturer is Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd. The liquid preparation method is as follows: Take 20 mg of Methotrexate tablets (8 in total, 2.5 mg/tablet) into a medical mortar, pour a small amount of carboxymethyl cellulose sodium (0.5%, w/v), and grind; After thorough grinding, dilute to 26.3 mL with sodium carboxymethyl cellulose (0.5%, w/v) to prepare a suspension with a concentration of 0.76 mg/mL, and store for future use.

1.1.2 Main reagents and equipment

Bovine Type II Collagen (Immunization Grade) (20022, Chondrex Company, USA); Freund's Complete Adjuvant CFA (5881, Sigma Company, USA; Freund's Incomplete Adjuvant IFA (F5506, Sigma Company, USA); BD AccurC6 Flow Cytometer and corresponding detection reagents are sourced from BD Company in the United States; and Rat Inflammatory Factor Kit (Suspension Chip Method Reagent) is purchased from Bio Rad Company in the United States; Hematoxylin dye solution (product number: ZH200304, China Google Biotech Co., Ltd.) and Eosin dye solution (product number: ZH202305, China Google Biotech Co., Ltd.).

Biooptical microscope was purchased from OLYMPUS ® (BX43 type, Japan), GENEX pipettes (10, 100, 200, and 1000 µ L) were purchased from Baide Laboratory Instruments (Suzhou) Co., Ltd. Bio-Plex 200 system was Luminex corporation, Austin, TX, USA), LEICA ® The automatic dyeing machine (AutoStainer XL model, Germany) and 1/10000 analytical balance were purchased from Mettler Company in the United States.

1.2 Animal Model Experiment

1.2.1 Rat preparation

Animal model experiment Sprague Dawley rats (SD rats), purchased from Guangdong Medical Experimental Animal Center, were raised and tested in its SPF level animal laboratory. The animal breeding room was kept at room temperature of 20–26 ℃, relative humidity of 40–70%, artificial lighting time of 12 hours/day, and automatic ventilation. The feed and drinking water for SD rats are both feed and purified water that meet the standards of the Guangdong Medical Experimental Animal Center. They are free to eat and can be freely consumed in drinking water bottles. Cage feeding (4 rats/box) was conducted, and individuals who were able to eat and drink normally were included in the experiment after one week of observation. Animal use license number: SYXK (Guangdong) 2018-0002; The ethical approval number is: 13202109-4. All animals were sacrificed with euthanasia method by 2%

For the animals experiments, according to the "Declaration of Helsinki of the World Medical Association", "Measures for Ethical Review of Biomedical Research Involving Humans" and other relevant domestic and foreign ethical norms, the ethics committee of Guangdong Medical Experimental Animal Center conducted an ethical review of this project. The ethics committee applies the simplified procedure, and only after reviewing the research proposal and other related materials submitted by the project applicant, considers that the project is and only in the project design and application, the protection of subjects involved in the research, and the risk-benefit ratio. Ethical issues such as assessments have been given sufficient attention and have been reasonably designed to meet ethical requirements. All efforts were made to minimize the number of rats used and to decrease their suffering. The animal experiments of this study were approved by the Institutional Animal Care and Use Committee of Guangdong Medical Experimental Animal Center (Approval No. 13202109-4).

1.2.2 CIA modeling, grouping, and gavage of SD rats

Male SD rats (48 rats) were randomly divided into a healthy group (8 rats) and a model group (40 rats). All SD rats underwent two measurements of the volume of their hind limbs and toes, recorded as day 0 (D0); Starting from the first day of modeling (D1), the modeling group was treated as follows: Using a syringe (Hamilton), SD rats in the model group were injected subcutaneously at the root of the tail, 2 cm in the leg direction, and 0.5 cm in the leg direction to establish a type II collagen induced arthritis (CIA) rat model (200 µ L/rat), the volume of both hind limbs and toes of SD rats was measured twice; On the fifteenth day (D15), booster modeling treatment was carried out. SD model rats continued to use this type of syringe. Starting from the subcutaneous position of the tail root, SD rats were injected with an injection 3 cm in the direction of the leg, and emulsifier (after emulsification with collagen Freund's incomplete adjuvant mixture, 200 µ L/rat) was injected at a depth of 1.5 cm; On the fourth week (28th day) after immunization, the incidence of SD rats was observed and the arthritis index score was recorded. Rats with a score of ≥ 1 were selected as successful models. 24 SD rats successfully selected for modeling were randomly divided into CIA model group, salivary lactobacillus group, and methotrexate group, with 8 rats as a group. After grouping was determined, all four groups of SD rats underwent two measurements of the volume of their hind limbs and toes;

After measurement, the healthy group was given bacterial culture medium by gavage (3 times/week, 1 mL/rat/time); The CIA model group was also given bacterial culture medium by gavage (3 times/week, 1 mL/rat/time); The Salivary Lactobacillus group was administered orally with Salivary Lactobacillus (3 times/week, 10¹⁰ CFU/rat/time); The methotrexate group was given methotrexate medication (once per week, 7.6 mg/kg, 10 mL/kg) by gavage, and each group was gavaged continuously for 4 weeks. At the end of the experiment, anesthetize the rats and collect blood for treatment, using a low-speed centrifuge 1006 ×g for 10 mins to obtain the upper serum, and place it at 4 ℃ for later use; Then execute and retain tissues such as the left ankle joint, knee joint, and spleen. The grouping and administration situation are shown in Table 1:

Table 1

Administration Situation of each group(♂)
Group	n	Test substance	Administration method	Dosage
Healthy group	8	Bacterial culture medium	Oral administration	1mL/rat/time, 3times/week
CIA model group	8	Bacterial culture medium		1mL/rat/time, 3times/week
Salivary Lactobacillus intervention group	8	Lactobacillus salivarius		10¹⁰CFU/rat/time, 1mL, 3times/week
Methotrexate group	8	Methotrexate		7.6 mg/kg, 1times/week

After the reperfusion period, the rats were euthanized immediately by exsanguination (total blood collection) under deep diethyl ether anesthesia. Eight rats of each group were anaesthetized using 2% isoflurane; the coeliac arterial blood was immediately isolated and centrifuged at 5000 rpm for 10 min, and the serum was used for immunological assay. Then, the rats were euthanized by exsanguination under 2% isoflurane anaesthesia.

1.3 Indicator observation and detection methods

1.3.1 Arthritis index score and measurement of foot thickness at onset

After continuous administration for four weeks, observe the redness and swelling of the four limbs and toe joints of rats in each group, and use the arthritis index scoring method [16] to evaluate the degree of joint inflammation in rats. Each rat is measured twice; and the thickness of the affected foot was measured using a vernier caliper.

1.3.2 Pathological Histological Observation Experiment

Hematoxylin eosin staining (HE) was employed to observe the pathological changes of the foot, whose operation method is to take the knee joint and fix it in polyformaldehyde (4%, V/V) for 24 hrs; After fixation, transfer to 70% alcohol (V/V) and continue with decalcification treatment; Then paraffin embedding, routine sectioning, and HE staining were performed, followed by observation and photography under an optical microscope.

1.3.3 Detection of cytokines in serum using liquid phase suspension chip

Using the Bio Plex 200 System suspension microbead chip platform, cytokine detection was performed on serum samples from each group to detect the expression of the following 8 cytokines: IL-1 β, IFN- γ, IL-10, IL-17, IL-2, IL-6, IL-8, and TNF-α by following the cytokine reagent kit manual from the corresponding suspension microbead chip platform.

1.3.4 Detection of Th17 cells and Treg cells

Take the separated spleen, then add pre-cooled PBS and store overnight at 2–8 ℃; the next day, grind it into individual spleen cells, and centrifuge it with 252×g for 10 minutes in a 4 ℃× Centrifuge, discard the supernatant; then add red blood cell lysate, and lyse on ice (0 ℃) for 1 hr. Then, perform trypan blue staining and cell counting; and adjust the cell concentration of the suspension into 1*10⁷ -3*10⁷ pieces/mL; stored until use.

1.3.4.1 Detection of Th17 cells

Take spleen single cell suspension and inoculate it into a 96-well plate; then adding 200 µ L cell culture medium (containing cell stimulator Brefeldin A), placed in a culture chamber (37 ℃ and 5% CO2) after cover plate stimulation for 5 hours to obtain stimulated spleen cells. After washing, the cell concentration was adjusted to 1.0 by cell counting × 107 pieces/mL, spare. Take 100 µ Place the spleen cell suspension at the above concentration in a flow cytometry tube, and then add anti rat CD16/CD32 antibodies (2 for each) µ L) Incubate on ice for 10 minutes; Add FITC labeled anti rat CD4 antibody (0.5 µ L) React in a dark room on ice for 25 minutes; Wash once with 1 mL PBS and centrifuge for 10 minutes (252 × g) Abandon and clear up afterwards; Add fixed solution (0.5 mL) and fix overnight at 4 ℃; 10 minutes on the second day (252 × g) After centrifugation, discard the supernatant and add intracellular staining and membrane breaking washing solution (1 mL) to wash once. Centrifuge for 10 minutes (252 × g) Abandon and clear up afterwards; Adding PE labeled anti rat IL-17A antibody (1.25 µ L) React on ice and avoid light for 0.5 hours, then wash with intracellular staining membrane breaking washing solution (1 mL), centrifuge for 10 minutes (252 × g) Abandon and clear up afterwards; Add PBS (300) to the precipitation µ L) Resuspension the cells and place them in a flow cytometry to detect the proportion of Th17 cells (Th17 cells contain IL-17 + and CD4 + labeled cell populations).

1.3.4.2 Detection of Treg cells

Take the spleen cell suspension above (100 µL) into a flow cytometry tube, and then add anti-rat CD16/CD32 antibodies (2 µL/each) into the tube, and incubate on ice for 10 mins. After the reaction, add the FITC labeled anti-rat CD4 antibody (0.5 µ L) and APC labeled anti-rat CD25 antibody (1.25 µL); then react on ice for 25 mins in dark; then centrifuge for 10 minutes (252 ×g) and discard the suspension after washing once with PBS. Next, add the prepared intracellular Fixation & Permeabilization buffer and incubate at room temperature for 45 mins in dark. Then centrifuge for 10 mins (252 ×g) and discard the supernatant after washed for once. And then add PE labeled anti-rat Foxp3 monoclonal antibody (2.5 µ L) and incubate on ice for 1 hr in dark. After the reaction, wash the reacted cells by adopting 1 mL of permeabilization solution; and centrifuge for 10 mins (252 ×g) with the supernatant discarded. Re-suspend the cells by using PBS (300 µ L). Then place these cells into a flow cytometer to detect the proportion of Treg cells (Treg cell populations containing Foxp3⁺, CD25⁺, and CD4⁺).

1.4 Statistical analysis

This study used SPSS 23.0 to process and statistically analyze the raw data, and all experimental data were presented in the form of x ̅ Represented in the form of ± s; Single factor analysis of variance was used for inter group comparisons, while t-test analysis was used for inter group comparisons, with P < 0.05 indicating significant differences.

2.1 Comparison of incidence of rheumatoid arthritis in rats

Compared with the healthy group, the arthritis index score and affected foot thickness of the CIA model group increased, and the difference was statistically significant (P < 0.05); Compared with the model group, the arthritis index score and foot thickness of the salivary lactobacillus group and methotrexate group were both reduced, and the differences were statistically significant (P < 0.05). There was no statistically significant difference in the arthritis index score and foot thickness between the methotrexate group and the salivary lactobacillus group. The results are shown in Table 2.

Table 2

Rheumatoid arthritis index score and affected foot thickness of rats in each group (x̅±s)
Group	n	Rheumatoid arthritis index score /score	Affected foot thickness /mm
Healthy group	8	0	4.11 ± 0.15
CIA model group	8	10.31 ± 0.79^a	6.92 ± 0.51^a
Salivary Lactobacillus intervention group	8	5.17 ± 0.92^a,b	4.89 ± 0.44^a,b
Methotrexate group	8	5.98 ± 0.86^a,b	4.95 ± 0.56^a,b
F value		258.352	57.748
P value		0.000	0.001
Note: a represents P ≤ 0.05 compared to the healthy group; b represents P ≤ 0.05 compared to the CIA model group.

2.2 Comparison of pathological and histological observations of the soles of rats in different groups (HE staining)

From Fig. 1, it could be found that HE staining of the soles of rats in each group showed no abnormal changes in the ankle joint tissue structure of healthy SD rats, all of which were regularly arranged ankle synovial cells without inflammatory cell infiltration or bone erosion (A). The SD rats in the model group had a large number of proliferative naked joint synovial cells arranged irregularly and disorderly, with distinct and increased levels. There was a large amount of inflammatory cell infiltration in the joint cavity, with extensive attachment of vascular pannus. The erosion and destruction of cartilage and bone were severe, and bone loss was severe. It was obvious that the ankle joint tissue structure had severe arthritis (B); The salivary lactobacillus group had mild arthritis in the ankle joint, with a small amount of proliferation, increased levels, and disordered arrangement of synovial cells in the ankle joint. A small amount of inflammatory cells infiltrated the joint cavity and formed a pannus, and a small amount of cartilage erosion and destruction (C); the methotrexate group had mild arthritis in the ankle joint (D).

2.3 Comparison of inflammatory factor levels in the serum of four groups of SD rats

Compared with the healthy group, the experimental group (CIA model group, salivary lactobacillus group, and methotrexate group) had IL-1 in the serum of rats β (T-values of 33.774, 23.523, 23.751), IFN- γ (T-values 27.886, 48.440, 56.423), IL-17 (T-values 51.212, 10.496, 18.678), IL-2 (T-values 77.151, 11.802, 25.157), IL-6 (T-values 42.959, 20.806/18.413), IL-8 (T-values 64.045, 6.470, 4.756), IL-10 (T-values 13.661, 2.558, 7.876), TNF- α The content of (T values of 37.974, 30.157, 23.073) significantly increased (P < 0.05); Compared with the CIA model group, the levels of various inflammatory factors in the salivary lactobacillus group and methotrexate group were significantly reduced (P < 0.05). There was no statistically significant difference in the inflammatory factors between the salivary lactobacillus group and the methotrexate group (P = 0.54, P > 0.05). The results are shown in Fig. 2.

2.4 The levels of Th17 and Treg cells and the ratio of Th17 and Treg in the spleen of each group

Compared with the healthy group, the level of Th17 cells in the spleen of rats in the CIA model group was significantly increased (P = 0.006 < 0.05), while the content of Treg cells was significantly reduced (P = 0.003 < 0.05), and the ratio of the two was significantly increased; Compared with the CIA model group, the Th17 cell levels in the salivary lactobacillus group and methotrexate group were significantly reduced (P < 0.05), while the Treg cell levels were significantly increased (P = 0.002 < 0.05), and the ratio of the two was also significantly increased (P = 0.004 < 0.05); Compared with the methotrexate group, there was no statistically significant difference in the content and ratio of Th17 cells and Treg cells in the salivary lactobacillus group (P = 0.54 > 0.05). The results are shown in Table 3.

Table 3

Content and ratio of Th17 cells and Treg cells in the spleen of rats in each group
Group	n	Th17(%)	Treg (%)	Th17/Treg
Healthy group	8	2.60 ± 0.08	24.88 ± 0.54	0.11 ± 0.01
CIA model group	8	5.84 ± 0.47^a	9.47 ± 0.30^a	0.62 ± 0.03^a
Salivary Lactobacillus intervention group	8	3.77 ± 0.14^{a, b}	13.04 ± 0.47^{a, b}	0.29 ± 0.11^{a, b}
Methotrexate group	8	3.41 ± 0.08^{a, b}	22.41 ± 0.33^{a, b}	0.15 ± 0.03^{a, b}
F value		238.918	2458.526	124.304
P value		0.000	0.000	0.000
Note: a represents P ≤ 0.05 compared to the healthy group; b represents P ≤ 0.05 compared to the CIA model group.

As a chronic autoimmune disease with unclear etiology, there is currently no specific treatment method for RA. Animal models have provided tremendous assistance for in-depth research on RA. Type II collagen is a protein that is isolated from the immune system, but under certain pathological conditions, it can be presented as an autoantigen and participate in autoimmune reactions [21]. A type II collagen induced arthritis model can be used, which can be induced by type II collagen from pigs, chickens, mice, cows, rats, or humans [22]. SD rats are a susceptible strain of CIA, and the advantage of this model is that it is similar to the clinical manifestations of human rheumatoid arthritis, with symmetrical joint involvement and significant changes in cellular and humoral immunity. It is an important model for studying the pathogenesis of human rheumatoid arthritis and drug efficacy. Based on this, this study used CIA susceptible SD rats as the research subjects, which are more representative. Changes in the composition of pulmonary, oral, and intestinal microbiota in preclinical and confirmed RA patients indicate that mucosal microbiota dysbiosis plays a role in the development and persistence of RA. Data from a mouse model of rheumatoid arthritis also supports this viewpoint. Several widely used treatment options for RA are associated with changes in gut microbiota, indicating that regulating gut microbiota and/or intestinal barrier function may be helpful in the treatment of RA [23–24].

As early as the 1980s and 1990s, scholars had paid attention to the changes in gut microbiota in human patients with rheumatoid arthritis. British scholar R After comparing the fecal samples of 25 RA patients with 25 normal control groups, Shinebaum found that the content of Clostridium perfringens in the feces of RA patients was significantly higher than that of the normal group, and the content of Escherichia coli was also higher than that of the control group. The content of Clostridium perfringens was correlated with the activity of rheumatoid arthritis, and the content of Clostridium perfringens was higher in patients with more severe disease activity [25]. This study selected one of the important members of the gut microbiota, Lactobacillus salivarius, as the treatment regimen. There is relatively little research on the effect of this probiotic on RA, which is also one of the innovative points of this article. This article uses Lactobacillus salivarius as the treatment plan in animal experiments to preliminarily explore the therapeutic effect of Lactobacillus salivarius on CIA. The results showed that compared with the healthy group, the CIA model group had a significant increase in arthritis index score and diseased foot thickness, while the Salivary Lactobacillus group had a significant decrease in arthritis index score and diseased foot thickness. Moreover, there was no statistically significant difference in therapeutic effect compared to the Methotrexate group, indicating that the two groups had similar therapeutic effects. HE staining of ankle joint slices indicates that intervention with Lactobacillus salivarius can improve joint pathological damage in CIA rats, similar to the function of the methotrexate group. The main manifestations are reduced synovial cell proliferation, inflammatory cell infiltration, and pannus formation, as well as reduced degree of cartilage and bone erosion and damage. Animal experiments have shown that Lactobacillus salivarius has anti-inflammatory effects and, like methotrexate, has significant therapeutic effects in the treatment of rheumatoid arthritis.

In addition, this experiment also analyzed the mechanism of improving RA by Lactobacillus salivarius from the perspectives of inflammatory factors, cellular immunity, and imbalance of cellular subpopulations. TH17 cells and Treg cells are both CD⁴⁺T cell subsets, and TH17 cells secrete IL-1 β、 IL-2, IL-6, IL-10, IL-17, and TNF- α Inflammatory factors promote the generation of osteoclasts, leading to bone destruction [26–27]; Treg cells, on the other hand, mainly inhibit the production of inflammatory cytokines by releasing anti-inflammatory factors or through cell contact, and have strong anti-inflammatory functions [28–31]. By comparing the cellular levels of the two, it can be seen that the CIA model group exhibits two distinct cellular imbalances, exhibiting a rat joint inflammatory response. After oral administration of Lactobacillus salivarius, the content of Th17 cells in the spleen was significantly reduced compared to the CIA model group, while the level of Treg cells was significantly increased, and the eight inflammatory factors IL-1 were also observed β、 IFN- γ、 IL-17, IL-2, IL-6, IL-8, IL-10, TNF- α The levels of bone and joint inflammation in rats were significantly downregulated, accompanied by a weakened inflammatory response. From this, it can be preliminarily inferred that Lactobacillus salivarius has an immunomodulatory effect, which is related to improving the immune imbalance of Th17 cells and Treg cells. The therapeutic effect is basically equivalent to methotrexate.

Lactobacillus salivarius has anti-inflammatory and immunomodulatory effects on CIA induced SD rats, and its mechanism of action may be to regulate the balance of Th17 cells and Treg cells in the spleen, inhibit the expression of inflammatory factors, and thus alleviate the occurrence and development of rheumatoid arthritis in CIA rats.

Statement

All authors declare that No conflict of interest exists. And we confirm that the study is reported in accordance with ARRIVE guidelines. And all experimental protocols were approved by the ethics committee of Guangdong Medical Experimental Animal Center. Meanwhile, all methods were carried out in accordance with relevant guidelines and regulations (GB/T 35892-2018).

Declaration of conflicting interest

All authors declare that No conflict of interest exists.

Funding

This study was supported by Dongguan Social Science and Technology Development (Key) Project (No.: 202050725001038)

Data availability

Data is provided within the manuscript or supplementary information files.

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Effects of Lactobacillus salivary on spleen T cells and inflammatory factors in a rat model of type II collagen induced arthritis Running title: Effects exploration of Lactobacillus salivary

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Abstract

Figures

Introduction

1. Materials and methods

1.1 Materials preparation

1.2 Animal Model Experiment

1.3 Indicator observation and detection methods

1.4 Statistical analysis

2. Results

2.1 Comparison of incidence of rheumatoid arthritis in rats

2.3 Comparison of inflammatory factor levels in the serum of four groups of SD rats

3. Discussion

4. Discussion

Declarations

References

Additional Declarations

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