Therapeutic Effects of Live Lactobacillus plantarum GKD7 in a Rat Model of Knee Osteoarthritis

Osteoarthritis (OA) is a painful, progressive chronic inflammatory disease marked by cartilage destruction. Certain synovial inflammatory cytokines, such as IL-1β and TNF-α, promote OA inflammation and pain. Lactobacillus spp. is a well-known probiotic with anti-inflammatory, analgesic, antioxidant, and antiosteoporotic properties. This study evaluated the therapeutic effects of a live L. plantarum strain (GKD7) in the anterior cruciate ligament transection (ACLT)-induced OA rat model. The results show that oral administration of live L. plantarum GKD7 improved weight-bearing asymmetry after ACLT surgery. Moreover, micro-computed tomography images and histopathological analysis show that oral live L. plantarum GKD7 improved subchondral bone architecture, protected articular cartilage against ACLT-induced damage, and reduced synovial inflammation. L. plantarum GKD7 also reduced IL-1β and TNF-α production in OA cartilage and synovium. Thus, orally administered live L. plantarum GKD7 appears to effectively slow the progression of OA.


Introduction
Osteoarthritis (OA) is a chronic inflammatory and degenerative joint disease accompanied by often debilitating pain [1]. MRI and micro-CT scans provide vital objective evidence for clinical diagnosis or experimental research of OA-related damage in synovial tissue, cartilage, and subchondral bone [2,3]. NSAIDs and corticosteroids are the mainstays of OA treatment for reducing OA pain, joint swelling, and stiffness [4,5], while joint replacement is considered to be the last therapeutic option for patients with severe joint pain or dysfunction that cannot be treated with pharmacotherapy [4,6]. Importantly, NSAIDs and corticosteroids have undesirable side effect profiles, some of which can be extremely serious [7]. Thus, safer and more efficient treatment strategies are needed for OA.
Reducing proinflammatory cytokine production (e.g., IL-1β and TNF-α are some of the most important therapeutic aims in OA [5], as these inflammatory mediators play important roles in the destructive changes in cartilage and modification to subchondral bone structural changes in OA [8,9]. The secretion of IL-1β and TNF-α from OA synovial fibroblasts and chondrocytes promote the synthesis of proteolytic enzymes that degrade joint extracellular matrices and thus drive OA, worsening the disease-related synovial inflammation, cartilage degeneration, and subchondral bone lesions [9][10][11]. In experimental OA, inhibiting or reducing pro-inflammatory expression suppresses joint degradation, highlighting the importance of such strategies for OA treatment [12,13].
Probiotic Lactobacillus spp. strains exhibit a wide variety of beneficial activities in human hosts, including reductions in inflammatory activity [14]. This study investigated the ways in which the L. plantarum GKD7 strain affects knee OA in rats subjected to ACLT surgery. Probiotics are considered safe for consumption due to their presence in many foods and their gut defense mechanism [15]. Proinflammatory cytokines such as IL-1β can be reduced by heat-killed L. plantarum L-137 in cardiac and adipose tissue in a rat model of metabolic syndrome, while L. rhamnosus GG can decrease TNF-α expression in lipopolysaccharide-activated murine macrophages [16,17]. L. salivarius UCC118 and L. plantarum WCFS1 have shown anti-arthritic activity in mice with collagen-induced arthritis, with evidence of reduced joint destruction and less proinflammatory cytokine activity [18]. Similarly, diverse Lactobacillus spp., including L. rhamnosus GG and L. rhamnosus LR-2, slow the progression of OA by reducing joint pain and inflammation [19,20]. Moreover, much clinical evidence attests to probiotics reducing intestinal damage and inflammation associated with OA disease and probiotics lessen pain severity and cartilage destruction in animal models of OA [19,21,22]. In our previous study, L. plantarum GKD7 reduced inflammatory cell infiltration in mice with aspirin-induced gastric injury [23]. We, therefore, speculated that L. plantarum GKD7 may slow OA progression by reducing disease-related inflammatory activity. This study examined the effects of orally administered live L. plantarum GKD7 upon joint inflammation, pain, swelling, and function in rats with ACLT-induced OA.

Preparation of L. plantarum GKD7
L. plantarum GKD7 isolated from Taiwanese pickles was cultured in de Man-Rogosa-Sharpe (MRS) medium for lactobacilli (Merck, Darmstadt, Germany) at 37 • C for 16 h. To prepare the L. plantarum GKD7 freeze-dried powder, 0.03% of seed culture was scaled-up in a 15-ton bioreactor with a 12-ton working volume in a culture medium containing 5% glucose, 2% yeast extract, 0.05% MgSO 4 , 0.1% K 2 HPO 4 , and 0.1% Tween 80. After 16 h of incubation at 37 • C, pellets of fermented bacteria were harvested by centrifugation, washed twice with reverse osmosis (RO) water then lyophilized with skim milk. The freeze-dried L. plantarum GKD7 powder contained approximately 5 × 10 11 CFU/g live bacteria, according to the plate count method.

OA Protocol
Eight-week-old male Sprague Dawley rats were supplied by LASCo Inc. (Taipei, Taiwan) and maintained in an animal center under the Institutional Animal Care and Use Committee (IACUC) Guidelines issued by China Medical University (CMU). The rats were randomly assigned to arthrotomy only (controls; n = 6), ACLT alone (n = 6), or ACLT + L. plantarum GKD7 treatment (n = 8). ACLT surgery followed the procedures described in previous studies [24,25]. Briefly, the rats were anesthetized with Zoletil 50 ® (Virbac, Carros, France) before undergoing an arthrotomy to expose the right knee joint, in which the ACL was cut by micro-scissors using surgical loupes. ACLT success was confirmed by the anterior drawer test. Starting from two days after surgery, the daily diet was supplemented with 1 mL RO water in all 3 study groups. L. plantarum GKD7 powder was suspended evenly in RO water and administered daily to each individual rat as an oral 100 mg/kg (5 × 10 10 CFU/kg) dose for 6 weeks in the ACLT + L. plantarum GKD7 group; the ACLT-only group and controls were each dosed daily with RO water alone. All rats were sacrificed by CO 2 on day 49 and the right hind knee samples were collected for micro-CT and pathological analysis, as well as immunohistochemistry (IHC) staining. All experimental procedures were approved by the IACUC of CMU (approval number: CMUIACUC-2021-291).

Weight-Bearing Testing of Hind Paws
The static weight-bearing incapacitance test (Bioseb, Paris, France) was performed every week to assess spontaneous pain and postural deficits. Each rat was placed in an angled plastic chamber and the hind paws were placed on separate sensors to measure betweenlimb differences in dynamic weight bearing (expressed as g) over a 10-s period [26]. The force value was calculated by the following equation: [Force = weight on left limb − weight on right limb]. Each experiment was repeated 3 times and the mean was recorded for each rat.

Histopathological Analysis
After undergoing micro-CT scanning, the right knee joints were prepared for hamatoxylin and eosin (H&E) staining, Safranin-O/Fast-green staining, or immunohistochemistry (IHC) staining. Briefly, the right knee joints were decalcified by 10% EDTA in phosphate-buffered saline and embedded into paraffin blocks for histology slices with H&E, Safranin-O/Fast-green, and IHC staining. Histopathological changes using an optical microscope, following our previously published procedures [27,[30][31][32]. The Osteoarthritis Research Society International (OARSI) histopathology grading system evaluated changes in structural cartilage from the medial tibial plateau (the weight-bearing area) [33,34], defining the grade of damage from 0 to 6 as the depth of OA progression into the cartilage and the stage of damage as the horizontal extent of cartilage damage from 0 to 4. The final score (grade × stage) ranges from 0 (normal cartilage) to 24 points (most advanced grade and most extensive stage) as in previous studies [33,34]. Cartilage degeneration was scored as 'none' to 'severe' (numerical values 0 to 5) [33,34], and surgery-induced inflammation in the synovial membrane was graded from 0 to 4 as described in our previous studies [35,36].

Statistical Analysis
All data are shown as the mean ± SD and analyzed by using Sigma Plot 12.0 software (Systat Software Inc., Berlin, Germany). The significant difference between the three groups was analyzed by using the unpaired two-tailed Student's t-test and one-way analysis of variance followed by Student-Newman-Keuls post hoc testing. A p-value of <0.05 was considered to be statistically significant.

Oral Live L. plantarum GKD7 Reduces Pain-Related Behavior without Affecting Body Weight
By week 6, all 3 study groups had steadily gained body weight from baseline, without any significant between-group differences (Figure 1). At week 1, measurements of asymmetry in weight-bearing posture did not differ significantly between the ACLT-only and ACLT + L. plantarum GKD7 groups (52.0 ± 3.7 g vs. 46.1 ± 6.0; p = 0.06). From week 1 onwards, ACLT-only rats exhibited severe asymmetry in weight-bearing posture, whereas pain-related behavior in the ACLT + L. plantarum GKD7 group was markedly improved from the second week onwards; by week 6, the asymmetry in weight-bearing behavior was approximately half that of the ACLT-only group (22.4 ± 5.0 g vs. 58.1 ± 6.7 g; p ≤ 0.05) and close to that of the control group (7.7 ± 2.8 g; p < 0.05) ( Figure 2). previous studies [35,36].

Statistical Analysis
All data are shown as the mean ± SD and analyzed by using Sigma P (Systat Software Inc., Berlin, Germany). The significant difference be groups was analyzed by using the unpaired two-tailed Student's t-test an ysis of variance followed by Student-Newman-Keuls post hoc testing. A was considered to be statistically significant.

Oral Live L. Plantarum GKD7 Reduces Pain-Related Behavior without Aff Weight
By week 6, all 3 study groups had steadily gained body weight from out any significant between-group differences (Figure 1). At week 1, m asymmetry in weight-bearing posture did not differ significantly betwee and ACLT + L. plantarum GKD7 groups (52.0 ± 3.7 g vs. 46.1 ± 6.0; p = 0.0 onwards, ACLT-only rats exhibited severe asymmetry in weight-b whereas pain-related behavior in the ACLT + L. plantarum GKD7 grou improved from the second week onwards; by week 6, the asymmetry in behavior was approximately half that of the ACLT-only group (22.4 ± 5.0 p ≤ 0.05) and close to that of the control group (7.7 ± 2.8 g; p < 0.05) ( Figur   Figure 1. Oral live L. plantarum GKD7 did not affect body weight in the OA rat model. Body weights of ACLT rats that did or did not receive oral live administration of live L. plantarum GKD7 were not significantly different from those of controls. Figure 1. Oral live L. plantarum GKD7 did not affect body weight in the OA rat model. Body w of ACLT rats that did or did not receive oral live administration of live L. plantarum GKD7 w significantly different from those of controls.

Figure 2.
Oral live L. plantarum GKD7 improved weight-bearing deficits after ACLT surgery oral administration of live L. plantarum GKD7 was associated with significantly less weight-b asymmetry after ACLT surgery compared with ACLT-only rats. * p < 0.05 vs. controls; # p < 0 the ACLT-only group.

Micro-CT Analysis Revealed Protective Effects of L. plantarum GKD7 in OA Bone
Coronal and transverse micro-CT images revealed marked subchondral bone the ACLT-only group compared with controls, whereas minimal loss was evident ACLT + L. plantarum GKD7-treated rats ( Figure 3A). BMD, BMC, BV/TV, BS/TV, and Tb.N measurements were all significantly smaller, while Tb.Sp measurements significantly larger, in the ACLT-only group compared with controls ( Figure 3Bcontrast, daily oral administration of live L. plantarum GKD7 was associated with s cant improvements in changes to subchondral bone induced by ACLT surgery (F 3B-H). Thus, L. plantarum GKD7 appears to reduce deleterious changes to knee joint ture induced by ACLT. Figure 2. Oral live L. plantarum GKD7 improved weight-bearing deficits after ACLT surgery. Daily oral administration of live L. plantarum GKD7 was associated with significantly less weight-bearing asymmetry after ACLT surgery compared with ACLT-only rats. * p < 0.05 vs. controls; # p < 0.05 vs. the ACLT-only group.

Micro-CT Analysis Revealed Protective Effects of L. plantarum GKD7 in OA Bone
Coronal and transverse micro-CT images revealed marked subchondral bone loss in the ACLT-only group compared with controls, whereas minimal loss was evident in the ACLT + L. plantarum GKD7-treated rats ( Figure 3A). BMD, BMC, BV/TV, BS/TV, Tb.Th, and Tb.N measurements were all significantly smaller, while Tb.Sp measurements were significantly larger, in the ACLT-only group compared with controls ( Figure 3B-H). In contrast, daily oral administration of live L. plantarum GKD7 was associated with significant improvements in changes to subchondral bone induced by ACLT surgery (Figure 3B-H). Thus, L. plantarum GKD7 appears to reduce deleterious changes to knee joint structure induced by ACLT.

Histopathological Analysis of L. plantarum GKD7 in OA Rats
Staining by H&E and Safranin-O/Fast Green revealed less injury (imaged were more intact) in articular cartilage samples from the controls and ACLT + L. plantarum GKD7 group compared with the minimal cartilage in the ACLT-only samples ( Figure 4A). ACLT-induced synovial inflammation (marked by arrowheads) and cartilage damage (arrows) were minimal in the ACLT + L. plantarum GKD7 samples, but very apparent in the ACLT-only samples ( Figure 4A). OARSI, cartilage, and synovium scores were significantly superior in the ACLT + L. plantarum GKD7 group compared with scores in the ACLT-only group, while the ACLT-only scores were significantly superior compared with the control group ( Figure 4B-D).
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Histopathological Analysis of L. plantarum GKD7 in OA Rats
Staining by H&E and Safranin-O/Fast Green revealed less injury (imaged were more intact) in articular cartilage samples from the controls and ACLT + L. plantarum GKD7 group compared with the minimal cartilage in the ACLT-only samples ( Figure 4A).

IHC Analysis of Proinflammatory Markers
As shown in Figures 5A and 6A, IL-1β and TNF-α levels were markedly upregulate in ACLT-only cartilage and synovium compared with control and L. plantarum GKD7 car tilage and synovium. The ACLT-only group had significantly higher IHC scores of cart lage and synovium compared with both the controls and the L. plantarum GKD7 group these values were significantly decreased by L. plantarum GKD7 when compared wit ACLT-only ( Figures 5B,C, and 6B,C).

IHC Analysis of Proinflammatory Markers
As shown in Figures 5A and 6A, IL-1β and TNF-α levels were markedly upregulated in ACLT-only cartilage and synovium compared with control and L. plantarum GKD7 cartilage and synovium. The ACLT-only group had significantly higher IHC scores of cartilage and synovium compared with both the controls and the L. plantarum GKD7 group; these values were significantly decreased by L. plantarum GKD7 when compared with ACLT-only ( Figures 5B,C and 6B,C).

Discussion
Proinflammatory cytokines have an important role in OA joint destruction [8,37], promoting damage in joint extracellular matrices and stimulating the progression of OA [9,10]. High IL-1β and TNF-α levels upregulate proteolytic enzymes, such as MMPs and ADAMTS family, which degrade the extracellular matrix [9,11]. Probiotics are well recognized for their anti-inflammatory properties [38,39] and previous research has highlighted the IL-1β and TNF-α cytokines as important targets in probiotic treatment strategies for arthritic diseases [19,40]. In particular, consumption of Lactobacillus spp. reduces proinflammatory cytokine production in experimental OA [19,41,42]. This was supported by our study results, as IL-1β and TNF-α levels in rat cartilage and synovium were markedly reduced by daily oral administration of live L. plantarum GKD7.
Coronal and transverse micro-CT imaging revealed minimal changes in subchondral bone architecture in the ACLT + L. plantarum GKD7 group, whereas there was marked subchondral bone loss in the ACLT-only group compared with controls. Subchondral bone lesions, including bone marrow edema and angiogenesis, are known to contribute to OA joint destruction [43,44], while IL-1β and TNF-α stimulate MMPs and ADAMTSs activity that subsequently worsens bone architecture, reflected by reductions in BMD, BMC and BV values, which promote OA disease progression [9,45]. The recent studies also indicated that subchondral bone lesions, including bone marrow edema and angiogenesis, also contribute to the OA joint destruction. Our micro-CT images revealed significant improvements in bone microarchitectural parameters of rats treated with live L. plantarum GKD7 following ACLT surgery compared with ACLT-only rats. In the L. plantarum GKD7 group, we observed significantly lower cartilage and synovium IHC scores, and also significantly reduced IL-1β and TNF-α levels, compared with the ACLT-only group. We, therefore, speculate that L. plantarum GKD7 ameliorates OA-induced changes in bone microarchitectural parameters by inhibiting proinflammatory levels in cartilage and synovial tissue. Thus, our data suggest that daily oral administration of live L. plantarum GKD7 can effectively reduce synovial inflammation and damage to bone architecture induced by ACLT surgery.
OA disease is accompanied by often severe pain [46]. In OA, the upregulation of TNF-α and IL-1β expression in the injured joint induces mechanical pain hypersensitivity by increasing the expression of nerve growth factor, which in turn increases the phosphorylation state of transient receptor potential vanilloid receptor 1 (TRPV1) [46][47][48], critical mediators of inflammatory pain signaling. Moreover, OA-induced disturbance of subchondral bone and cartilage destruction results in abnormal load-bearing in the joints and is an important contributor to chronic pain during non-weight-bearing activities [44,49]. This abnormal load-bearing causes the release of multiple signaling mediators such as nerve growth factor, neuropeptide substance P, and neurokinin-1 in the osteochondral area, resulting in persistent nociception stimulation and the characteristic OA pain [44]. Thus, reducing the expression of TNF-α and IL-1β and improving the cartilage destruction and "abnormal" loading within the subchondral bone are important targets of OA treatment [1,43,45]. In our results, ACLT surgery significantly increased levels of TNF-α and IL-1β expression, led to the development of weight-bearing deficits in rat hind legs, and structural damage in articular cartilage, compared with changes observed in the sham-operated group. Daily oral administration of live L. plantarum GKD7 significantly reduced these ACLT-induced effects, which suggests that this probiotic strain can effectively ameliorate OA pain.

Conclusions
Consumption of live L. plantarum GKD7 may improve OA disease progression and associated joint pain by reducing levels of IL-1β and TNF-α in OA joints and by improving weight-bearing asymmetry. We suggest that L. plantarum GKD7 may be a useful strategy for improving the signs and symptoms of OA. Informed Consent Statement: Not applicable.

Data Availability Statement:
The raw data for this study are available from the corresponding authors.