Quinic acid alleviates inflammatory responses and oxidative stress in Freund’s complete adjuvant-induced arthritic rat model and associated risk factors of atherosclerosis

Quinic acid (CAS Number: 77-95-2) with 98% purity was acquired from Shanghai Macklin Biochemical Co., Ltd. and stored according to instructions given on the label. Distilled water was used to dissolve the drug and make a solution. Standard drug methotrexate was obtained from Paramedic Laboratories (PVT) LTD, Lahore.

Using the simple random sampling method, 35 albino rats, weighing 160–210 g, 6–8 weeks old of either sex was acquired from University of Veterinary and Animal Sciences (UVAS), Lahore, and retained in the Lahore College for Women University’s animal house for 7 days until they were accustomed to their new surroundings. The rats were kept individually in well-ventilated cages (n = 5). Rats were kept under standard conditions such as 23 ± 3 °C, a 12-h alternating dark and light cycle, and humidity maintained between 30 and 70%. The rats were provided with regular diet and ample supply of water. All obligatory precautionary measures for safety of animals were carried out (Zeb et al. 2024).

The authorization for animal studies was acquired via the Institutional Research Ethical Committee of Lahore College for Women University, Lahore (ORIC/LCWU/41-24).

For induction of polyarthritis, 0.15 mL FCA (heat-killed Mycobacterium, ZOKEYO®) was injected subcutaneously into the left hind paws, specifically targeting the subplantar region of rats of all the groups except the V. Ctrl group rats at day 0 (Hannan et al. 2023).

For this study, rats were categorized into seven groups; each group contained five rats. After development of polyarthritis, the treatment was commenced on the 8th day and lasted until the 22nd day. Based on available in vivo acute toxicity data (Arya et al. 2014) and previously published literature, the treatment drug quinic acid was administered orally to the treatments groups at different dose levels such as 25, 50, and 100 mg/kg body weight (Ghasemi‐Dehnoo et al. 2023), corresponding to the low, medium, and high dose, respectively. One treatment group received combination treatment that was 100 mg/kg body weight of QA orally and methotrexate 0.75 mg/kg body weight intraperitoneally (Saleem et al. 2022). Each rat was killed on the 23rd day following the induction of polyarthritis. The timeline is illustrated in Fig. 1.

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Group-I: vehicle control group (V. Ctrl)

Healthy rats were given distilled water as a vehicle orally.

Group-II: arthritic control (A. Ctrl)

Arthritic control rats received distilled water as a vehicle for treating arthritis induced by FCA.

Group-III: rReference drug control (RDMTX)

Arthritic rats were given reference drug methotrexate (0.75 mg/kg b. w., IP) weekly for 15 days, beginning on the 8th day and extending to the 22nd day after arthritis was induced (Abdel-Maged et al. 2019).

Group-IV: Low-dose QA group (LDQA 25 mg/kg)

Arthritic rats received a low dose of quinic acid (25 mg/kg b. w.) orally using oral gavage for 15 days, beginning on the 8th day and extending to the 22nd day after arthritis was induced.

Group-V: medium-dose QA group (MDQA 50 mg/kg)

Group-VI: high-dose QA group (HDQA 100 mg/kg)

Arthritic rats were given a high dose of quinic acid (100 mg/kg b. w.) orally using oral gavage for 15 days, beginning on the 8th day and extending to the 22nd day after arthritis was induced (Ghasemi‐Dehnoo et al. 2023).

Group-VII: high dose + reference drug group (HDQA + RDMTX)

Arthritic rats of this group received combination therapy. The rats received a high dose of quinic acid (100 mg/kg b. w. orally) treatment and reference drug methotrexate (0.75 mg/kg IP). The HDQA 100 mg/kg was given orally using oral gavage daily for a period of 15 days, while RDMTX was given IP once a week for 15 days, beginning on the 8th day and extending to the 22nd day after arthritis was induced.

Using an electronic weighing balance (D446410976 Shimadzu Corporation, Japan), the body weight was determined on 0, 8th, 13th, 18^th, and 23rd days. To determine the percentage change in body weight, the following formula was used.where \(wx\) represents the rat’s weight on day x, while \(wo\) refers to its weight on 0 day (Kaur et al. 2023).

Paw thickness (mm) was measured using a digital vernier caliper on 0 day before FCA induction and then on the 8th, 13th, 18th, and 23rd days. The percentage paw edema inhibition was computed by the formula:where \(\text{Eo}\) denotes the paw size at 0 day (prior to FCA induction), \(\text{Et}\) refers to the paw size at each consequent day starting from the 13th day, and \(\left(Et-Eo\right)\) shows the paw edema (Gul et al. 2023).

Based on visual criteria, the arthritic score was assessed at the 0, 8th, 13th, 18^th, and 23rd day. The severity of the disease was graded from o to a 4 score, where a score of 0 signifies no pathological changes, while a score of 1–4 indicated minimal paw erythema and swelling, mild paw swelling and erythema, moderate erythema and swelling of the entire paw, and severe swelling and deformity of the paw, respectively (Saleem et al. 2022).

Joint stiffness was assessed using a scale from 0 to 2. Rats were delicately handled from behind in the left hand's palm and securely grasped on the back. The right hand's fingers were then used to bend and extend the knee (almost five times). The restraint of motion in both bending and extension (once for every direction) was the basis of the scale. Score 0 was assigned to no restrictions, score 1 referred to complete ankle joint restriction in bending or extension, and score 2 denoted complete range of motion restriction in both bending and extension of the ankle joint in rats (Kaur et al. 2023; Waseem et al. 2025).

On the 23rd day, ketamine/xylazine cocktail anesthesia was delivered via the IP route at a ratio of 40 mg/kg to 10 mg/kg to euthanize the animals (Parveen et al. 2023). The blood sample from each rat was collected in vacutainers via cardiac puncture. Blood was drawn and injected into an EDTA tube for hematology and PCR analysis (Gul et al. 2023). On the other hand, the blood for the serum separation was collected in a gel clot activator tube for the analysis of lipid profile, antioxidant enzymes, ADMA, and homocysteine level. For a histological evaluation, the rats were killed to get the left ankle joints and heart tissues. The joints and heart tissues were kept in tightly sealed containers that contained 10% neutral buffered formalin solution.

After collection of blood in gel clot activator tubes, the tubes were left undisturbed over a period of 15–30 min at room temperature to allow clotting. The serum was obtained by centrifugation for 10 min at 4 °C and 3000 rpm (1020D centurion Scientific, UK) (Saeed et al. 2022). The serum samples were transferred to Eppendorf tubes and kept in a freezer (Ultra Low Temp Freezer U410 Premium, New Brunswick Scientific, USA) at − 80 °C until biochemical analysis (Ahmed et al. 2015).

Hematological indicators such as Hb content, red blood cell count, platelet, and leukocyte count were determined in blood samples from rats using an automatic hematology analyzer (Celltac α MEK-6500K Hematology Analyzer, Japan) (Haroon et al. 2024).

The serum was used to measure the lipid profile of rats. The TC, high-density lipoprotein, total triglycerides, very low-density lipoproteins, and low-density lipoproteins were estimated by means of a biochemistry analyzer (SBA-733PLU, China) (Malik et al. 2022).

The concentration of ADMA in rat’s serum sample was measured using ADMA ELISA kit for rats (LOT no. 00501, Ideal Medical Technology Co., Ltd. Shanghai, China). The procedure was carried out following the instruction provided by the kit’s manufacturer. The optical density was recorded at 450 nm by a microplate reader (BIOBASE-EL 10A, China) within minutes. The standard curve of known ADMA concentrations was used to determine the ADMA concentration in rat serum samples and the results were depicted in µmol/L (Anwar et al. 2025).

Homocysteine concentration in rat’s sera samples was measured via Rat Homocysteine ELISA kit (CSB-E13376r, CUSABIO®). The procedure was performed according to the manufacturer’s protocol provided with the kit. Optical density was recorded at 450 nm in 5 min. Through the use of a standard curve, the homocysteine concentration in sera of rats was calculated and the results were represented in nmol/mL (Anwar et al. 2025).

For determining the SOD activity, equal quantities of 0.2 mM pyrogallol solution (50 µL) and serum sample (50 µL) were mixed with 1500 µL phosphate buffer. Following the 10-s induction period, absorbance will be measured at 325 nm every 30 s for 5 min via an ELIZA reader (BIOBASE-EL 10A, China), which is the wavelength at which the radicals absorb light most effectively (Khan et al. 2020, 2024).

CAT activity was determined by a slight modification in the previous method. By mixing 50 µL of serum, 50 µL of 30 mM H₂O₂ (pH 7.0), and 100 µL of 0.1 M PBS, the reaction mixture was prepared. Next, an ELISA reader (BIOBASE-EL 10A, China) was used to measure the absorbance values every 5 s at 240 nm. The activity of CAT was determined using the extinction coefficient of H₂O₂ (0.071 mmol⁴ cm⁻¹) and the results were expressed in U/mL (Zafar et al. 2021).

The MDA level was measured by slight modification of previous methods. 0.375% thiobarbituric acid (TBA), 15% trichloroacetic acid (TCA), and 0.25 N hydrochloric acid (HCl) were added together to create a TBA solution for the measurement of MDA level (Bahrami et al. 2016). 150 µL of TBA solution was mixed with 50 µL of serum sample. After thoroughly combining the solutions, it was placed in a bath of boiling water for 15 min and quickly cooled in an ice bath. The supernatant was collected after centrifuged at 3000 rpm for 10 min (Eppendorf centrifuge 5417R). A microplate reader (BIOBASE-EL 10A, China) was used to measure supernatant absorbance at a wavelength of 532 nm (Shahbaz et al. 2024; Zeb et al. 2024). The MDA level was measured in nmol/ m L (Ansari et al. 2019).

By slight modifications to the Ellman's method, the concentration of GSH in serum was measured. Using this approach, 30 min of centrifugation at 3000 rpm (Eppendorf centrifuge 5417R) was used to precipitate equal quantities of serum and 10% trichloroacetic acid solution (50 µL). Following that, the clear supernatant was taken out and 200 µL of PBS was added. Later on, 25 µL of (5,5′-dithiobis-(2-nitrobenzoic acid) reagent (Lot #.C12350703) was added. After incubating the samples at room temperature for 15 min, thiol groups that were present in GSH reacted with DTNB to break its disulfide bond and produce 2-nitro-5-thiobenzoate (TNB₂ ⁻) ions. The presence of TNB₂ ⁻ ions can be detected by an appearance of yellow color. Using an ELISA reader (BIOBASE-EL 10A, China), the samples' absorbance was recorded at 412 nm wavelength. The standard curve of known glutathione concentrations was employed to estimate the GSH amounts in rat serum samples and the results were expressed in nmol/mL (Mukhtiar et al. 2012; Saeed et al. 2022; Shakir et al. 2023).

As nitric oxide (NO) is unstable, it is difficult to measure the production of NO; instead, we measure the level of nitrite (NO₂⁻), one of the stable by-products of NO, which is a molecule that is stable and non-volatile in contrast to NO (Gravandi et al. 2023). The nitric oxide (NO) production can result from oxidative stress and can be assessed by measuring nitrite levels. Griess reagent was used to measure the nitrite level. After evenly combining the serum sample (50 μL) and Griess reagent (50 μL), the resulting mixture was left to incubate for 10 min, and the absorbance at 546 nm was recorded with an ELIZA reader (BIOBASE-EL 10A, China). The concentration of nitrite content in the sample was estimated based on the standard curve of known concentrations and the results were reported in nmol/ mL (Bais et al. 2015).

The levels of inflammation markers such as CST, IL-16, IL-1β, DDAH1, and TNF-α in the blood of rat was determined by qPCR. Using Multi-type Sample RNA Extraction-Purification Kit (Cat no. S1006E, Sansure Biotech Inc., China), RNA was extracted from blood samples in accordance with the manufacturer’s standard protocol. The quantification of RNA samples was carried out using a NANODROP 2000 spectrophotometer (Skanit RE 4.1, Thermo Scientific). ThermoFisrt cDNA kit (HRP013 100 T ZOKEYO, China) was used to reverse transcribe RNA samples to generate cDNA, with a minimum RNA input concentration of 10 ng/uL (Fatima et al. 2024). SYBR Select Master Mix (Cat No. 4472903, ZOKEYO, China) was used to achieve real-time expression of targeted genes using cDNA as template and duplicates of the relevant primers (10 µM each) (synbio technologies). Using the universal thermal cycling conditions, PCR reactions were run on SLAN-96P Real-Time, PCR System (Sansure Biotech Inc. China). After the software generates a cycle of thresholds (Ct values), the Ct values of samples were compared to those of control samples and control samples containing housekeeping genes (GAPDH). The fold expression of a gene change was calculated using the Livak method (ΔΔCt) (Siddique et al. 2024). The primer sequences employed in the study are presented in Table 1 and have been chosen from previous literature and synthesized commercially (Synbio Technologies).

At the termination of the study, research animals were killed on the 23rd day for histopathological evaluation of ankle joint tissues. After the ankle joints were separated, they were fixed for 36 h in 10% neutral buffered formalin. They were decalcified with decalcifying solution (ethylenediamine tetraacetic acid, hydrochloric acid, and potassium and sodium tartrate) for 48 h. The tissues that were collected required further processing before being embedded in 5 μm-thick paraffin blocks (Naz et al. 2020). Following embedding, each paw's thin histological slices were carefully processed and affixed on glass. H&E staining was subsequently applied to enable one to see their morphology under a research microscope (LFM-B10 Labtron Equipment Ltd UK). Hematoxylin imparts a violet or blue color to cellular nuclei, whereas eosin imparts red or pink color to extracellular and cytoplasmic structures. The H&E staining makes it easier to thoroughly examine pannus development, inflammatory cell infiltration, and bone erosion in rat paws, and enabling comprehensive histopathological examinations (Djehiche et al. 2024). The histopathological analysis, for the bone erosion, cartilage erosion, synovial hyperplasia, inflammation, pannus formation, and vascular congestion was assessed by scores of 0–3, where score 0 represented no pathological changes, 1 mild changes, 2 moderate changes, and 3 severe damage (Maarouf et al. 2024; Munir et al. 2021).

The heart tissues were collected and quickly rinsed off with ice-cold saline to get rid of all the blood, and it was then preserved in a 10% buffered neutral formalin solution (Pullaiah et al. 2021). Following that, it was properly dehydrated in 100% ethanol and embedded in standard paraffin blocks. These paraffin blocks were used to prepare slices of heart tissue that were 4–5 µm thick, placed on a glass slides, and then stained using hematoxylin–eosin. Stained sections were evaluated via a research microscope (LFM-B10 Labtron Equipment Ltd UK) that was computer connected to take tissue photomicrographs (Olorundare et al. 2020). The parameters of the heart's histopathological assessment such as inflammation, hemorrhage, and vascular congestion were assessed by scores of 0–3, where score 0 represented no pathological changes, 1 represented mild changes, 2 represented moderate changes, and 3 represented severe damage (Akşit et al. 2023; Ali et al. 2023).

Statistical analysis of the data was performed with the help of GraphPad Prism software. The data was displayed as mean ± SD. One-way analysis of variance (ANOVA) was applied. Tukey’s post hoc test was used for multiple comparison. *: represents the comparison of treatment groups with arthritic rat group. *, **, *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

V. Ctrl rats exhibited a gain in body weights throughout the research (13.64% increase by 23rd day), whereas A. Ctrl rats body weights displayed a sharp decline, reaching a largest drop of nearly 19.41% (p < 0.001) by the 23rd day (Table 2). Interestingly, QA delivered at a dose of 100 mg/kg when given in combination with RDMTX substantially inhibited the marked drop in body weight over a course of disease up to the 13th day (0.58%; p < 0.01) and during the recovery phase from day 13 to day 23 (9.04%; p < 0.001). Animals receiving various doses of QA exhibited a pronounced regain in body weight by day 23 when compared to the A. Ctrl group. Treatment with RDMTX also prohibited the drop in body weight. The rats in this group exhibited an increase of 6.56% (p < 0.001) relative to the A. Ctrl group. Conclusively, rats treated with HDQA 100 mg/kg + RDMTX exhibited maximum recovery during the 23rd day study period.

All treatment groups exhibited a % paw edema inhibition relative to the A. Ctrl group rats by the 23rd day as given in Table 3. Among these treatment groups, the HDQA 100 mg/kg combined with RDMTX exhibited the most significant percentage paw edema inhibition (66.7%) and reduction in mean paw thickness (4.704 ± 0.244 mm; p < 0.001) at day 23 as compared to arthritic control rats (6.752 ± 0.296 mm). Furthermore, the rats treated with different doses of QA (100 mg/kg,50 mg/kg, and 25 mg/kg) and RDMTX also had % inhibition of paw edema (59.31%,43.77%,33.55%, and 27.98%, respectively) and decrease in mean paw thickness (5.346 ± 0.399 mm; p < 0.001, 5.678 ± 0.291 mm; p < 0.001, 5.878 ± 0.421 mm; p < 0.01, and 4.922 ± 0.467 mm; p < 0.001 correspondingly) compared to the A. Ctrl group. However, the extent of this inhibition and reduction in mean paw size was less pronounced when it was compared with the group in which QA was administered in combination with the standard drug.

The arthritic score was increased in all groups excluding the V. Ctrl group on the 8th day, as no disease was introduced in the V. Ctrl group, and the readings of this group’s rats were taken as 0. On day 13, 18, and 23, the arthritic score was gradually decreased in all the treatment groups in contrast to the A. Ctrl group, whereas an increase was observed in the arthritic control group throughout the study. On the 23rd day, among all treatment group rats, the group in which HDQA100mg/kg was combined with RDMTX exhibited the most substantial reduction (p < 0.001) in arthritic score when compared to A. Ctrl rats, as shown in Fig. 2.

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Joint stiffness score (Fig. 2) was increased across all groups except the V. Ctrl group rats on the 8th day. In the V. Ctrl group, the readings were taken as 0, as no disease was induced. Joint stiffness score was gradually increased in the A. Ctrl group as compared to the V. Ctrl group, while treatment with various doses of QA (25 mg/kg, 50 mg, and 100 mg) and RDMTX decreased the joint stiffness score on the 13th, 18th, and 23rd days, but this drop was not statistically significant in contrast to the A. Ctrl group rats. HDQA100mg/kg combined with RDMTX was significantly most effective in reducing the joint stiffness score (p < 0.001) relative to A. Ctrl rats by the 23rd day.