Introduction

Rheumatoid arthritis is a persistent autoimmune inflammatory disease that manifests as a symmetrical polyarthritis characterized by joint swelling, pain, and stiffness1. Approximately 70% of people living with rheumatoid arthritis are women, and 55% are older than 55 years2. Pathological examinations reveal involvement of both joint and extra-articular organs, including eye, skin, nervous system, heart, kidney, lung, and digestive system3,4. In the recurrence of rheumatoid arthritis, patients experience general symptoms such as loss of appetite, anaemia, and listlessness, which affect the quality of life5. The aetiology of rheumatoid arthritis is still unclear; however, female sex and environmental and genetic factors are known risk factors in the development of rheumatoid arthritis6.

Treatment approaches for rheumatoid arthritis generally consist of disease-modifying anti-rheumatic drugs7, non-steroidal anti-inflammatory drugs8, corticosteroids9, analgesics10, and biologic agents11. However, these treatments have many adverse effects, such as lymphoma, liver problems, and increased risk of infections12,13.

Ganoderma Lucidum (GL) is a fungus used for more than 2000 years for therapeutic purposes like chronic bronchitis, hepatitis, hyperglycemia, and leukopenia14. GL, which comprises two main compounds (triterpene and polysaccharide), is believed to be accountable for many pharmacological activities15. Ganoderic acid A (GAA) is one of the most abundant triterpenoids in GL, and has been proved to possess a wide range of beneficial health effects.16. GAA may prevent the release of cellular histamine and strengthen the function of various digestive system organs. In addition, GAA is against hyperlipidemia and inflammation and exhibits hepatic protection17,18.

Cytokines that indicate the presence of inflammation, such as TNF-α, IL-6, and IL-1β, can be measured to monitor the presence of arthritis and the effectiveness of treatment5. It is also reported that nuclear NF-κB is active in inflammation-producing pathways, both in pathogenesis and progression, especially in relation to genes, and therefore will be found high in the presence of inflammation5. In autoimmune and chronic inflammatory disorders such as rheumatoid arthritis, the liver plays an important role in the modulation of the immune response and in detoxification processes during treatment. AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are reliable enzymes in measuring the health status of the liver19.

The present study aims to investigate the therapeutic effect of GAA, on inflammation and pain sensation in the rheumatoid arthritis model. The effects of GAA were investigated by triggering autoimmune response in the rheumatoid arthritis model through collagen and adjuvant.

Materials and methods

Animals

We purchased 40 Balb/c male mice (4–6 weeks old, weighing 15–18 g) from Sakarya University Experimental Medicine Application And Research Center (Sakarya, Türkiye). They were all maintained in an animal lab under standard conditions with access to food and water ad libitum, and their health status was checked regularly. All animal experimental procedures were approved by Sakarya University Animal Experiments Local Ethic Committee (No: 16/ June 7th, 2023). The study was conducted under the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations.

Drugs and reagents

Type II collagen (C7806), CFA (F5881), and IFA (F5506) were purchased from Sigma Aldrich Co. (St. Louis, USA). Methanol (Tekkim, Türkiye, TK.120320.01001) and GAA (purity ≥ 98%, Tauto Biotech Shanghai, China), used as a solvent for GAA, were purchased. Acetic acid (100056, Merck, Germany) was purchased.

Experimental design

Mice were randomly divided into five groups (n = mice/group): Control (C), Acetic acid (AA), rheumatoid arthritis model (RA), and Low GAA (LGA) and High GAA groups (HGA) as treatment groups.

Group C received physiologic saline (PS) intra-articularly (ia) via a 10 µl Hamilton syringe into the left knee joint. After ia injection, 100 µl PS was injected subcutaneously (sc) into the dorsal region of mice. AA group received 0.1 M AA ia via a 10 µl Hamilton syringe into the left knee joint. After ia injection, 100 µl PS was injected sc into the dorsal region of mice. After 21 days, the second immunization was implemented in the mice in the same group at the same dose and with the same method20,21. All ia injections were performed under anesthesia.

A final concentration of 2 mg/ml Type II collagen was emulsified into 0.1 M AA for the first immunisation to induce rheumatoid arthritis. RA and GAA groups were administered collagen emulsion via 10 µl Hamilton syringe ia into the left knee joint. Immediately after the injection of collagen emulsion, 100 µl of CFA was injected sc into the dorsal region of mice. After 21 days, the second immunization was implemented with collagen emulsion in the mice in the same group at the same dose and with the same method. However, mice were injected with IFA instead of CFA.

GAA was dissolved in 50%MeOH22,23 and volume completed with PS. LGA group received 20 mg/kg/day GAA and HGA group received 40 mg/kg/day by gavage for 9 days. Groups C, AA and RA also received similar volumes of PS via gavage for 9 days. At the end of the experiment, all rats were euthanized by the intraperitoneal injection of 120 mg/kg sodium pentobarbital (see graphical abstract in Fig. 1). Blood was collected by cardiac puncture for analysis5.

Fig. 1
Fig. 1
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Overview of the study design: Graphical representation of the experimental groups of rats considered to study the effect of GAA at 20 mg/kg and 40 mg/kg in RA. The rats were divided into five groups and injections were made on days 1 and 22. From day 22, GAA was given to the LGA and HGA groups at 20 mg/kg and 40 mg/kg, respectively.

Follow-up parameters

Body weight

The weight of mice in each group was evaluated and recorded every other day.

Arthritis score

The severity of arthritis was scored from 0 to 4 in each extremity according to the arthritis score24. The arthritis score was evaluated once every three days before the experiment (Fig. 2).

Fig. 2
Fig. 2
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The picture shows arthritis assessments of the upper extremity (top) and lower extremity (bottom). (a = No oedema or swelling; b = Mild oedema and limited erythema; c = Mild oedema and erythema from ankle to tarsal bone; d = Moderate oedema and erythema from ankle to tarsal bone; e = Oedema and erythema from the ankle to the entire leg).

Knee temperature

The skin temperature of the knee was measured above the patella using an infrared thermometer (Infrared Thermometer IR988, INFRARED). The measurements were made once every three days25.

Knee circumference

The circumferences of both knees were measured and evaluated with a calliper (0–150 mm calliper, JKTOOL). Measurements were made before the experiment, the second immunization, and the sacrifice. A calliper at the joint level measured the knee’s anteroposterior and mediolateral diameters (mm).

Lower extremity index

The method applied by Chen et al. for the assessment of the lower limb index was modified26. During the sacrifice process, the dissected lower limbs were weighed on a precision scale after being separated from the muscles. The weighing result was then divided by the body weight to obtain the lower limb index (mg/g).

Liver index

Organ coefficient gives information about experimental animals’ functional status and histopathology. Therefore, the liver index was calculated using the formula organ weight/body weight × 100%27.

Behavioural and locomotor measurements

The behavioural assessment scale of pain

Knee joint postures were measured every 2 days to evaluate spontaneous pain throughout the experimental period28.

Gait test

The hind paws of the mice were dipped in ink, and they were allowed to walk 60 cm on white paper before the experiment29, in the 2nd week, and before the sacrifice (Fig. 3).

Fig. 3
Fig. 3
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Gait test. (a) Normal footprint; (b) partial footprint (no heel); (c) fingers only; (d) no single footprint; (e) total absence of footprint.

Tail flick test

Before the experiment, in the second week, and before sacrifice, the tails of the mice, which were gently restrained at their necks, were positioned with the central part of the tail placed 0.5 cm away from the proximal end of the heat source. The moment the tail moved was recorded30.

Hot plate test

Before the experiment, in the second week, and before sacrifice, mice placed on a hot plate device set at 55 ± 0.5 °C were observed for moments when they licked and/or jumped using their paws24.

Locomotor activity test

Before the experiment and the sacrifice, the locomotor activity cabinet system used Ferreira’s method to evaluate the activity. The animals were placed in the centre of the cabinet, and behaviours were recorded for 5 min using the Ethovision-XT video tracking system. The locomotor activity was evaluated by measuring the total distance taken in the cabinet. Each mouse was individually allowed to roam in a 40 × 40 cm area for 5 min, and the distance taken by the mice during this period (cm) was recorded31.

Biochemical evaluation

In the Biochemistry laboratory of Sakarya University Training and Research Hospital, liver samples stored at − 80 °C for tissue homogenization were cut into small pieces by removing connective tissues on ice. Samples were weighed and placed in glass tubes containing cold phosphate buffer (pH 7.4, 50 mmol/L) to a final concentration of 100 mg tissue/mL. The homogenization process was performed on ice in a mechanical homogenizer (Isolab, Laborgerate GmbH, Germany). The resulting homogenate was separated from debris and other particles by centrifugation at 10,000g, 4 °C, for 10 min. All parameters were studied from supernatants obtained after centrifugation. Commercial kits in the study were processed in accordance with the kit protocol. NFkB (BT Lab, Cat. No: E1350Mo), ALT (BT lab, Cat no: E0494Mo), IL-6 (BT lab, Cat. No. E0049Mo), AST (BT lab, Cat. No: E0329Mo), TNF (BT lab, E0117Mo) levels were analyzed spectrophotometrically (Biotek ELX-800 Washer, USA; Biotek ELX50 Reader Instruments, Winooski, VT, USA).

Histopathologic evaluation

After sacrifice, the left extremities of the mice were removed, and the knee joints were prepared for histopathological examination. They were immersed in 10% buffered neutral formaldehyde solution for light microscope examination and left for 48 h for fixation. Decalcification of cartilage-bone plugs was performed using 5% formic acid (pH = 2). The samples were immersed in continuously stirred decalcifying solutions at room temperature for 48 h32. Tissue samples fixed with buffered neutral formaldehyde were processed through the following tissue processing steps in a semi-closed system automatic tissue processor after trimming and then washed in spring water after 48 h. After completing the tissue processing steps, they were embedded in paraffin. Sections of 4 µm thickness were obtained with a microtome from the prepared paraffin blocks. These sections were placed on positively charged slides and left at room temperature to dry until the staining process. They were then ready for Hematoxylin and Eosin (H&E) staining. After completing the H&E staining steps, the slides were coverslipped with Entellan and prepared for examination under the microscope. The tissues were scored using a light microscope according to the arthritis severity abbreviated by SLM26 and according to the morphological changes scale as abbreviated by MLM26,33.

Statistical analysis

The obtained data were determined as mean ± standard deviation (SD). SPSS version 22 and R 4.4.2 programs were used for statistical analysis. The figures were created using “ggplot2” package. Data were tested for normality with Shapiro–Wilk.The differences between groups were analyzed using the Kruskal–Wallis test. One-way ANOVA and Posthoc Tukey tests were used for evaluation among groups. p < 0.05 values were considered statistically significant.

Results

Follow-up results

Effects of GAA on body weight of mice

Compared with the C group, the body weight of mice in the RA group was significantly decreased in the RA group on the 18th, 24th, 26th, and 28th days (p < 0.05). However, there was no significant difference between RA and GA groups (p ≥ 0.05, Table 1).

Table 1 Comparison of body weight of groups.
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Effects of GAA on clinical arthritis score in mice

The clinical arthritis score was significantly higher in the RA group than in the C group at all measurement days during the experiment (p < 0.05). Even though the RA group’s arthritis score was no different from that of the LGA group, the RA group’s arthritis scores were significantly higher than those of the HGA group on the 30th day (p ≥ 0.05, p < 0.05 respectively). Compared with the AA group’s arthritis score, the RA group was significantly higher on the 13th, 16th, 18th, and 30th days (p < 0.05). However, there was no difference between the LGA and HGA groups (Fig. 3) (p ≥ 0.05).

Effects of GAA on joint temperature

The RA group’s right and left knee temperatures were significantly warmer than the C group on the 30th day (p < 0.05). The RA group’s left knee temperature was significantly warmer than the LGA group’s on the 6th, 24th, and 30th days (p < 0.05). In addition, the RA group’s right knee temperature was warmer than the LGA group on the 12th, 18th, 21st, and 24th days (p < 0.05). The knee temperature of the RA group was warmer than the HGA group on the 12th and 24th days (p ≥ 0.05). There was no significant difference between GAA groups (Figs. 4, 5) (p ≥ 0.05).

Fig. 4
Fig. 4
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The clinical arthritis score is shown in groups by day. Each group is represented by distinct colors in the figure. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A groups.

Fig. 5
Fig. 5
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The knee joint temperature is shown in groups by day. Each day is represented by distinct colors in the figure. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A groups. Day0-L before experiment left knee, Day0 R before experiment right knee, Day30 L 30th day left knee, Day 30 R 30th day right knee.

Effects of GAA on knee circumference

Mediolateral (ML) and anteroposterior (AP) circumferences were also measured. Before the second induction of RA and sacrifice, the joint circumference measurements of group C were significantly narrower than those of group RA (p < 0.05). Before the sacrification, the LGA group’s left and right ML circumferences were significantly narrower than the RA group (p < 0.05). The HGA group’s left AP and ML circumferences were significantly narrower than the RA group (p < 0.05). However, there was no difference between GAA groups (Fig. 6) (p ≥ 0.05).

Fig. 6
Fig. 6
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The knee joint circumferences are shown in groups by day. Each group is represented by distinct colors in the figure. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A groups, ML mediolateral, AP anteroposterior.

Effects of GAA on lower extremity index

The C group had a significantly lower extremity index than the RA group. No significance was found in other group comparisons.

Effects of GAA on liver ındex

When the liver index values of the groups were compared, the index value of the RA group was significantly higher than the Control and AA groups (p < 0.05). The index value of the RA group was significantly decreased in LGA and HGA groups (Table 2) (p < 0.05).

Table 2 Comparison of liver index of groups.
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Behavioural and locomotor measurements

Effects of GAA on the behavioural assessment scale of pain

The RA group scored significantly higher than the C group on the experiment’s 6th, 26th, 28th, and last day (p < 0.05). Before the sacrification, the RA group had significantly higher scores than the GAA group (p < 0.05). However, there was no difference between GAA groups (p ≥ 0.05).

Effects of GAA on gait test

The RA group scored significantly higher than the C group in the second week and before the sacrification (p < 0.05). Before the sacrification, the RA group was significantly higher than the LGA group (p < 0.05). However, there was no difference between GAA groups (p ≥ 0.05).

Effects of GAA on tail flick test

The time between placing the tail on the heat source and its withdrawal was considered the reaction time. It was found that, before sacrifice, the reaction time of the RA group was significantly less than the C group (p < 0.05). No statistically significant difference was found when comparing groups C and AA (p ≥ 0.05). Before sacrifice, it was also found that the RA group had significantly less reaction time than the LGA group (p < 0.05). Similarly, a significant difference was observed between groups RA and HGA before sacrifice, with group RA having a significantly less reaction time (p < 0.05). No statistically significant difference was found between GAA groups (p ≥ 0.05).

Effect of GAA on hot plate

The HGA group has a significantly higher reaction time than the RA group before the sacrification (p < 0.05).

Effects of GAA on locomotor activity test

When comparing the groups C and RA, it was found that, before sacrifice, the distance taken by group C was statistically significantly higher than that of the group RA (p < 0.05).

Biochemical evaluation results

Biochemical levels of AST, ALT, IL-6, TNF-α, and NFkB are shown in Fig. 7.

Fig. 7
Fig. 7
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The biochemical parameters are shown in groups. Each group is represented by distinct colors in the figure. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A groups, AST aspartate aminotransferase, ALT alanine aminotransferase, IL-6 ınterleukin-6, TNF-α tumor necrosis factor-α, NFKβ nuclear factor kappa B.

AST levels: The RA group had significantly higher AST levels than the C, LGA, and HGA groups (p < 0.05).

IL-6 levels: The RA, AA and LGA groups scored significantly higher IL-6 levels than the C group (p < 0.05). The RA group also had a significantly higher score than the HGA group (p < 0.05).

TNF-α levels: The RA group had significantly higher TNF-α levels than the C and HGA groups (p < 0.05). The AA group had significantly higher scores than the C and HGA groups (p < 0.05).

NFkB Levels: The AA and LGA groups had significantly higher NFkB levels than the HGA group (p < 0.05).

ALT Levels: ALT levels didn’t statically change in any groups (p > 0.05).

Histopathologic evaluation results

When comparing the C and RA groups, it was found that the arthritis and morphological evaluation scores of the RA group were significantly higher (p < 0.05). The RA group had significantly higher scores in both arthritis score and morphological evaluation than the LGA group (p < 0.05). It was found that the data of the RA group were significantly higher in both arthritis and morphological evaluation scores than the HGA group (p < 0.05). When comparing the LGA and HGA groups, the HGA group had significantly lower scores in both arthritis score and morphological evaluation compared to the LGA group (p < 0.05) (Figs. 8 and 9).

Fig. 8
Fig. 8
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Histopathological arthritis evaluation in groups. Each group is represented by distinct colors in the figure. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A, SLM evaluation of arthritis severity in light microscopy, MLM evaluation of arthritic morphological changes in light microscopy.

Fig. 9
Fig. 9
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Microscopic image of the knee joint of the experimental groups stained with H&E: Black star: area of deteriorated cartilage tissue, black arrowhead: tissues growing in the joint space, Black triangle: areas of thinned cartilage and areas of inflammation; Black arrow: synovial tissue and inflammatory cells, Black star: area of deteriorated cartilage tissue, black arrowhead: tissues growing in the joint space. Scale bar: 200x. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A.

When comparing the experimental groups according to the liver sections examined (Fig. 10), no significant differences were observed between the groups according to the Suzuki scores (p > 0.05). In the LGA and HGA groups, blood accumulation in the vena portae and portal areas and blood flow and occasional congestion in areas close to these areas were observed. Cell necrosis and vacuolization areas were almost non-existent in all groups.

Fig. 10
Fig. 10
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Histopathological findings in all studied groups.Liver sections were examined at 100 × magnification and 50 scale bar. No difference was observed between the experimental groups according to the Suzuki parameters. C control, AA acetic acid, RA rheumatoid arthritis, LGA low ganoderic acid A, HGA high ganoderic acid A.

Discussion

In this study, we aimed to investigate the effect of GAA contained in Ganoderma Lucidum on arthritis in Balb/c mice, in which an experimental rheumatoid arthritis model was established using functional and histopathological methods. The results showed that the arthritis score of the RA group was higher than the other groups, and the arthritis score decreased in the GAA groups due to decreased edema and inflammation. GAA was also seen to reduce pain in treatment groups. Histopathologically, it was observed that it reduced the damage to joint cartilage and bone structures. This study was the first to investigate GAA’s functional, histopathological healing and analgesic effects on rheumatoid arthritis together.

A clinical arthritis score is used to grade arthritis, and the degree of arthritis increases as oedema and erythema increase in body areas affected by arthritis28. Our study found that the RA group had a significantly higher arthritis score than the C group on all days of measurement. When the C and AA groups were compared, it was found that the AA group had a statistically significantly higher arthritis score. In the histological findings were damaged areas in the cartilage tissue, areas where the bone tissue was dense and the cartilage tissue was thinned, and inflammatory cells were observed in some places in the AA group. This suggests that acetic acid can be used to create an arthritis model for the first time in the literature.

GAA, the most crucial component of triterpenoids, has been shown to have many pharmacological effects, such as anti-inflammatory, regulating and protecting liver functions, and has been reported as a potential therapeutic drug on intervertebral disc degeneration34. Our study observed that clinical arthritis scores decreased statistically significantly in the HGA group on the 9th day of treatment. In the study published in 20205, an experimental rheumatoid arthritis model using collagen and CFA at 1 g/L and GAA was applied to the treatment groups at 20 mg/kg and 40 mg/kg, respectively, for four weeks. When looking at the arthritis score, it was reported that the arthritis score of the RA group increased significantly. There was no statistical difference between the GAA and RA groups before the GAA application. Still, significant decreases were observed in the arthritis score in the GAA groups after GAA application. We think the significant difference in arthritis scores after GAA application in both GAA groups in this study is due to the extended period of GAA treatment. The article of Meng et al.35 investigated the anti-rheumatoid activity and possible mechanisms of GL polysaccharide peptide (GLPP) on the Collagen induced arthritis (CIA) model with a mixture of Type II collagen and CFA in rats. The results showed that the arthritis score in the GLPP (35 days/day intragastric) groups was significantly lower than the CIA group. Another study published recently36 aimed to elucidate the anti-rheumatoid effects on CIA model (type II collagen and CFA) mice treated with GL spores (GLS) oil twice a week for seven weeks. The arthritis score of the GLS oil group was shown to be significantly lower than that of the CIA group. These studies have shown that Ganoderma lucidum is effective in reducing arthritis levels, as in our study.

When the histopathological evaluation was examined, the RA group’s scores were significantly higher. When the GAA groups were compared with RA, the scores were statistically significantly lower in the treatment groups. In the RA group, thickening and edema were observed in the synovial tissues, while in the LGA group, the synovial tissues were of normal thickness. However, small degraded areas in the cartilage tissue and occasional thickened bone areas were observed. Normal joint cartilage and bone structure were observed in the HGA group. In the CIA model performed with GAA at doses of 20 mg/kg and 40 mg/kg, similar to our study, it was shown that high doses of GAA prevented the proliferation of synovial tissue in the joint to different degrees in the first weeks and reduced monocytes and lymphocytes to different degrees. Similar levels of improvement were observed in both doses from the 4th week onwards. These study data support our dose-dependent results. A study using the CIA model was also planned at similar doses5.

In our study, we observed an increase in knee temperature in the RA group and a significant decrease in both knees in the GAA groups, indicating the effect of GAA in our experiment. However, no study in the literature created a rheumatoid arthritis model that analyzes knee temperature and monitors its change in GAA. Our study filled this gap in the literature by showing that the increase in knee temperatures caused by RA was significantly reduced in the GAA groups. In a study37, the joint surface temperature was found to be significantly higher in the group created with heat-killed mycobacterium tuberculosis suspended in liquid paraffin to create an adjuvant-induced arthritis model in rats than in the control group. An increase in knee joint circumference, another inflammation marker, indicates an increase in oedema. Our study found that the RA group had a higher joint circumference. A 2016 study showing that CFA increased oedema25 reported that the joint circumference of the CFA group increased compared to the C group in rats in which monoarthritis was induced with CFA. In another study38, a single dose of 0.1 ml CFA in rats found significantly higher paw oedema in the CFA group than in the control group. However, Phadke et al. suggest that type II collagen plays a role in the chronicization of adjuvant-induced arthritis39. This method, which we also applied, appears to be an advantage of CIA over adjuvant-induced arthritis. In our study, joint circumferences were found to be lower in both treatment groups, in which we evaluated the oedema-reducing effect of GAA compared to the RA group. Our result demonstrated that GAA significantly reduced the circumference of the knee joint and this change was similar in both doses. The literature shows that the decrease in knee joint circumference indicates a decrease in inflammation25. Oedema has been measured in studies using many different methods in addition to calipers. Oedema was measured using the water volume method in GAA administration, which was given for four weeks in rats. The rheumatoid arthritis model was created by administering collagen and CFA5. Swelling of the toes began to increase in the CIA group at weeks 1 and 2 compared to group C. At week 3, swelling of the toes was reduced in the 40 mg/kg GAA group compared to the CIA group. At week 4, swelling of the toes was reduced in the 20 mg/kg GAA group compared to the CIA group. A significant reduction in the toe swelling was noted at week 6 in the 20 mg/kg and 40 mg/kg GAA groups. In this respect, our study showed that increased knee joint circumference caused by RA was significantly reduced in the GAA groups. In a study revealing the effect of GLPP on the CIA model35, it was found that in the CIA group, paw thickness increased significantly after the second immunization, reached the highest level on the 43rd day, and GLPP peptide application from the 38th day caused significant decreases in paw thickness. In this respect, it seems to be compatible with our study. In another study40 in which the analgesic and anti- rheumatoid arthritis effects of GL and San Mio San supplements were examined in the CFA-induced rheumatoid arthritis model, monoarthritis was created with 125 µl CFA applied to a single knee. This supplement was given at a dose of 50 mg/kg/day orally or intraperitoneally for seven days before induction with CFA and seven days after induction. The fact that this supplement containing GL reduces oedema, cartilage erosion and hyperemia in the rheumatoid arthritis model created with CFA supports our study. It has been reported that both intraperitoneal and oral administration reduces arthritis parameters such as oedema and hyperemia in the inflamed knee. The gavage application we prefer is non-invasive and does not harm living things compared to intraperitoneal applications. In this respect, it makes our work more advantageous.

Weight loss due to bone destruction, muscle loss and weakness is observed in rheumatoid arthritis41. In a study5 in which an arthritis model was created in rats by administering bovine type II collagen and CFA, GAA was applied for four weeks. In terms of body weight, there was a statistically significant lower body weight in the RA group compared to the C group, and this supported our study. However, this study has shown statistically significantly higher body weights were reported in the GAA groups compared to the RA group, but no significant difference was found in our study. This may be due to the difference in the animal type used and the duration of the GAA application. In another study, the GLPP application was applied for 35 days in rats with the rheumatoid arthritis model using the CIA model35. In this respect, weight gain was significantly less in the CIA model group than in the C group due to inflammation, immobilization, pain and fever, which is consistent with our study. In addition, GLPP has been shown to reverse weight loss. The CIA model is similar to our study in that it causes a decrease in weight, but the fact that GLPP reduces this decrease is due to the difference in the active ingredient.

It reduces weight bearing on the extremity of the mouse as a result of pain due to rheumatoid arthritis. In the behavioural assessment of pain, the weight bearing that develops due to pain decreases as the score increases. It was found that the RA group had statistically significantly higher behavioural values of pain. When groups C and AA were compared, it was found for the first time in literature that the AA group was statistically significantly higher in terms of behavioural values of pain. This supports the hypothesis that it can be used in the arthritis model.When the AA and RA groups were compared, it was found that the RA group was statistically significantly higher. When the RA group and treatment groups were compared, it was found to be significantly lower in the treatment groups. In a study35 revealing the effect of GLPP in the CIA model, they calculated the weight transfer capacity by calculating the average paw pressure and average paw area. We measured weight-bearing capacity by behavioural assessment of pain and gait assessment. They reported that paw pressure and paw area returned to their initial state significantly in the GLPP groups compared to the CIA group. In this respect, the literature supports our study.

An increase in the gait score indicates a decrease in weight-bearing capacity. In our study, the gait scores of the RA group were found to be statistically significantly higher. When the RA group and treatment groups were compared, it was found that the gait score of the RA group was significantly higher. In a study that used the CIA model and created an RA model and investigated the treatment effectiveness of Withania somnifera, commonly known as Ashwagandha29, it was reported that the gait score increased significantly in the CIA group compared to the C group on the 20th and 45th days. In this respect, our study is compatible with the experimental model in terms of walking assessment parameters. Since there is no publication in the literature evaluating the behavioural assessment of pain and walking score on the weight-bearing capacity of GAA in the rheumatoid arthritis model created using the CIA model, our study contributed to the literature in this respect.

In the Tail Flick test, the time between placing the tail on the heat source and withdrawing the tail is called reaction time, and its increase was interpreted as an increase in the analgesic effect30. In our study, the reaction time of the RA group was found to decrease significantly before sacrification. It was also found that the reaction time of the AA group was significantly shorter before the sacrification. And the reaction time of the RA group was found to be significantly shorter than the treatment groups. In the literature42, a study in which a rheumatoid arthritis model was created using the CIA model showed that the reaction time in the RA group was lower. This shows that the CIA model causes pain. Another study43 showed that the anti-nociceptive activity of GAA was higher than the acetylsalicylic group.

In the hot plate test, the action time was recorded after the mouse was placed on the hot plate, and the time between when the mice licked their front paws and/or jumped and the increase in action time shows that the analgesic effect is more remarkable. Our study observed that HGA groups showed a better anaesthesia effect in the hot plate test. A study published in 202336 examined the effects of GLS oil on the CIA model. Type II collagen and 4 mg/ml CFA were injected into the tail. The hot plate test demonstrated a significant analgesic effect of GLS oil 49 days after induction. It has been reported that the duration of the hot plate test was prolonged in the treatment group compared to the control group; that is, the reaction time increased, which revealed the analgesic effect. No study was found in the literature showing the analgesic effect of GAA using the tail flick test and hot plate test.

In the locomotor activity test, the increase in the distance travelled by mice is used to measure immobilization caused by rheumatoid arthritis. When the C and RA groups were compared, it was found that the distance taken by the C group before the sacrification was statistically significantly higher than the RA group. However, it has been shown that GAA application has no significant effect on immobilization. There is no study in the literature that measures the mobility of GAA with the locomotor activity test in the CIA model.

The lower extremity index was used to measure oedema. When the C and RA groups were compared, the RA group’s index was significantly higher. No statistically significant difference was shown when RA, LGA, and HGA groups were compared, but our study evaluating the effect of GAA on the lower extremity index is unique as it is the first study in the literature.

Li et al.44 conducted a double-blind, randomized, placebo-controlled study on RA patients measuring the safety and effectiveness of GL and San Miao San supplements. The supplement was observed to have an analgesic effect, and the pain score and the patient’s global score improved. However, the CD4 + /CD8 + /natural killer cell/B lymphocyte ratio did not change between groups. This study stated that GL and San Miao San had analgesic effects that were safe and well tolerated in patients with active rheumatoid arthritis. However, it has not been shown to have a significant antioxidant, antiinflammatory or immunomodulatory effect. We think this is because the most effective dose of Ganoderma Lucidum is unknown.

Although the effects of GAA on rheumatoid arthritis have been studied molecularly in the literature, its functional effects have not been fully revealed. Our study fills this gap in the literature. There are limited studies5,45 in the literature investigating the effects of GAA on arthritis. In one of these, Cao et al.5 reported that 4-week GAA treatment significantly reduced TNF-α, IL-6 and IL-1β levels in serum and synovium in rats in which the rheumatoid arthritis model was created using collagen and CFA. In another, in an in vivo model in which osteoarthritis was created by medial meniscus destabilization, it was revealed that metallopeptidase 13 could improve osteoarthritis by regulating the RANKL/osteoprotegrin ratio to inhibit the inhibition of secretion, and GAA could alleviate the pathology of medial meniscus destabilization45.

In our study, where ALT levels were not statistically different in any group, AST levels produced results consistent with the literature. AST levels were significantly higher in the RA group than in the C, LGA and HGA groups. A study showed that the liver index of mice that was impaired by alcohol intake was improved by oral administration of GAA, which prevented abnormal elevation of AST and ALT19. GAA significantly protected the liver against alcohol-induced excessive lipid accumulation and pathological changes in that study19.

Modern medicine believes that one of the pathogenesis of rheumatoid arthritis is closely related to various cytokines such as IL-6 and TNF-α. The therapeutic purpose of rheumatoid arthritis should be to reduce the inflammatory reactions5. In our study, the fact that the TNF-α and IL-6 levels of the RA group were higher than those of the control group indicates that inflammation was induced significantly. The fact that the TNF-α and IL-6 levels of the HGA group were significantly lower than the RA group also suggests that GAA has an anti-inflammatory effect. Cao et al.'s study also emphasized that 4-week GAA treatment reduced the levels of TNF-α and IL-6 in the serum and synovium of rats undergoing rheumatoid arthritis model5.

The production of NF-κB, which is active in animal models and the synovial tissue of patients with rheumatoid arthritis46, plays a role in the joint inflammation of rheumatoid arthritis47. In our study, NFkB levels in the HGA group were significantly higher than in the AA and LGA groups. This is consistent with the literature since NFkB levels are expected to increase during inflammation.

Conclusions

In conclusion, we focused on a new therapeutic agent, that is safe with fewer side effects, as the frequency of autoimmune diseases is increasing annually, that brings with it other complex autoimmune diseases and comorbidities, we see rheumatoid arthritis as the disease we encounter most frequently, and the difficulty of existing treatments due to side effects. These results revealed that the effects of GAA, the most potent ingredient of the mushroom known as Ganoderma Lucidum, which has been used for a long time and plays an essential role in Traditional Chinese Medicine as a therapeutic agent since its effect on rheumatoid arthritis is little known. This study showed that GAA may be effective because of its anti-inflammatory, antirheumatic, and analgesic properties.

Highlights

Rheumatoid arthritis is an autoimmune disease with no definitive treatment and requires combined pharmacotherapy to manage the disease.

Our study examined the anti-inflammatory, anti-rheumatic analgesic effects of Ganoderic Acid A in an experimental rheumatoid arthritis model in a dose-dependent manner. In this respect, it fills the gap in the literature.

Our study also examines the effects of Ganoderic Acid A on the liver in the control and Rheumatoid arthritis groups, filling the gap in the literature.