**3. Results**

#### *3.1. SNO, but Not HA, Attenuates Knee Joint Pain and Inflammatory Swelling in ACLT* + *MMx-Induced OA Rats*

Immediately after ACLT + MMx surgery, rats were assigned to di fferent treatment groups as described in Figure 1A. Following the surgery, in the OA-control group, we found that ACLT + MMx induced a constant and gradually increasing knee width as a result of progressive knee joint inflammation. In contrast, a preventive and long-term oral administration of SNO reduced the OA-induced knee joint swelling at 2 weeks post-treatment and eventually achieved a di fference of approximately 27.5% (OA-SNO: 1.89 ± 0.06 vs. OA-control: 2.61 ± 0.05 mm) at week 12. However, such knee reduction was not evident in the HA-treated group (OA-HA: 2.48 ± 0.07 mm vs. OA-control: 2.61 ± 0.05 mm) (Figure 2A). With an oral SNO (daily) added to the HA treatment as an adjuvant supplement since the 2nd week, we started to observe the reduction in knee width from 4th to 12th weeks post-surgery in OA-SNOHA rats, (Figure 2B). At the end, we found a 20.6% reduction in the knee width compared to that of the OA-control group (OA-SNOHA: 2.07 ± 0.10 vs. OA-control: 2.61 ± 0.05 mm).

**Figure 2.** Joint swelling measurement of the OA rats. ACLT + MMx was performed to induce knee OA at week 0, and different treatments were given according to the experimental design. ( **A**) Biweekly knee width measurements of the OA-control, OA-HA (hyaluronic acid), and OA-SNO (shea nut oil) rats. (**B**) Biweekly knee width measurements of the OA-control, OA-HA and OA-SNOHA (hyaluronic acid + shea nut oil) rats. The data are presented as the Δ knee width (mm), and the values are expressed as the mean ± SEM. Two-way ANOVA and Sidaks's multiple comparisons test were used to analyze the data. # *p* < 0.05, ##/\*\* *p* < 0.01, ### *p* < 0.001.

We used the weight-bearing test to assess pain behavior during OA progression (Figure 3A). Similar to our previous report, we found that ACLT + MMx-induced OA elicited a constant change in weight-bearing asymmetry compared to that of the sham-OP group, which only presented acute pain in the first few weeks as a result of the surgical procedure. The preventive and daily supplementation of SNO reduced the pain behavior up to 87.9% compared to that of the untreated OA-control group (OA-SNO: 5.22 ± 2.37 g vs. OA-control: 43.21 ± 6.93 g). However, HA alone did not yield any significant reduction in pain during our biweekly weight-bearing measurements; at the end, HA monotherapy yielded only a 25.3% reduction in treated rats compared to OA-control rats (OA-HA: 32.33 ± 5.90 g vs. OA-control: 43.21 ± 6.93 g). When the two treatments were given together since the 2nd week (Figure 3B), the OA-SNOHA rats showed a maximum reduction in pain (71.6%) at the 10th week as compared to O-control (OA-SNOHA: 15.51 ± 6.13 g vs. OA-control: 54.58 ± 7.90 g), and a 75.9% reduction when compared to the rats treated with OA-HA alone (OA-SNOHA: 15.51 ± 6.13 g vs. OA-HA: 64.37 ± 8.67 g).

**Figure 3.** Weight bearing test of the OA rats. ACLT + MMx was performed to induce knee OA at week 0, and different treatments were given according to the experimental design. (**A**) Biweekly weight-bearing measurements of the sham-OP, OA-control, OA-HA, and OA-SNO rats. (**B**) Biweekly weight-bearing measurements of the sham-OP, OA-control, OA-HA, and OA-SNOHA rats. The data are presented as the Δ Force (g) and expressed as the mean ± SEM and two-way ANOVA, and Sidaks's multiple comparisons test were used to analyze the data. # *p* < 0.05, ##/\*\* *p* < 0.01, aaa/###/\*\*\* *p* < 0.001.

Next, we evaluated the synovial reaction as a microscopic sign of internal inflammation of the joint. In the sham-operated joint, we found a single layer of synovial lining cells without proliferation of the subsynovial tissue or vascular changes (Figure 4, sham-OP). As a result of the ACLT + MMx, the OA-control joint showed an increased thickness of both synovial lining cells, and the subsynovial tissue contained extensive extracellular matrix. Along with hypervascularity of the subsynovial area, this result suggested a chronic synovial reaction as result of surgically-induced OA (Figure 4, OA-control). Upon IAHA treatment (Figure 4, OA-HA), the histological findings showed a relative reduction in the synovial reaction compared to that of the OA-control joint, which suggested attenuation of OA with the lubricating action of IAHA. Moreover, with the addition of SNO supplementation to IAHA (Figure 4, OA-SNOHA) or oral SNO alone (Figure 4, OA-SNO), the two modalities of treatment offered better anti-inflammatory protection, as shown by the amelioration of the synovial reaction with less synovial proliferation and subsynovial thickness.

#### *3.2. SNO, HA Alone, and HA Plus SNO, O*ff*ered Significant Improvement in Cartilage Integrity in Knee OA Rats*

The cartilage deterioration was evaluated at 12th week post-surgery using the OARSI score system as shown in Table 1 and Figure 5. It is clearly noted that ACLT + MMx (OA-control) caused significant extensive matrix loss and a deformed cartilage surface, while the sham-OP cartilage showed a thin and smooth surface and preserved cartilage integrity. The quantitative data of the OA-HA, OA-SNO, and OA-SNOHA rats, showed a significant attenuation of cartilage matrix loss (specially in surface 0% and mild-depth 50% level) compared to that of the OA-control rats.

**Figure 4.** Synovium reaction of the OA joint. Representative sections of the medial femoral condyle joint capsule of the knee joints from the sham-OP, OA-control, OA-HA, OA-SNO, and OA-SNOHA rats at week 12 post-surgery were shown. ST = subsynovial tissue; S = synovial lining cells. Scale bar = 200 μm.



All operated knee joints were collected at 12 weeks post-surgery and were processed with toluidine/fast green staining for evaluation using OARSI's parameters. The bold text shows the value with statistical significance. Asterisk denote the statistical examination of each group in comparison with OA-control using Student's *t*-test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001

The cartilage degeneration score showed that ACLT + MMx induced a total lesion score of 6.208 ± 0.408, with the inside zone (2.708 ± 0.195) being the most affected region. The treatment with IAHA injection or combined SNO showed a significantly lower total degeneration score (5.000 ± 0.371 and 4.791 ± 0.307) than the OA-control (6.208 ± 0.408). In addition, we found that those rescues were observed primary in the inside zone, where all treatments (HA, SNO, and SNOHA) showed significant protective effects (1.833 ± 0.214, 1.833 ± 0.177, and 1.583 ± 0.133) compared to OA-control (2.708 ± 0.195).

Next, we quantified the extension (measured in width, mm) of the cartilage degeneration. The parameters are further subclassed as total (any type of degenerative change) or significant (seriously compromised, 50% of chondrocytes are absent or necrotic) and as the zonal depth ratio. Both OA-HA and combined treatment OA-SNOHA led to a significant attenuation of the 3 parameters, while OA-SNO more specifically decreased the significant cartilage degeneration width and zonal depth ratio. In summary, the oral SNO treatment alone significantly improved 5 of the 10 parameters we measured, while both IAHA and SNOHA treatment yielded significant ameliorations of 7 out of 10 parameters.

**Figure 5.** Representative section of medial tibial cartilage from each treatment (OA-HA, OA-SNO, and OA-SNOHA) and control (sham-OP, OA-control) were shown. Scale bar = 500 μm.

#### *3.3. Metabolic Profiling of OA Rats Receiving Daily Oral Supplementation of SNO*

Firstly, we found no difference of body weight between sham-OP and OA-control, but a significantly, but minor increase of body weight was detected in the OA-HA group at the 12th week (Figure 1A). On the other hand, we found gradual and minor reduction of body weight in OA rats supplemented with oral SNO, including those receiving IAHA at the same time (Figure 1B). We previously demonstrated that long-term oral SNO supplementation in OA rats decreased body weight and blood TG level without altering the blood aspartate transaminase (AST), blood urea nitrogen (BUN), and cholesterol levels [21]. Here, we compared the full metabolic profile at the 4th, 8th, and 12th weeks. Similarly, we found no alteration of uric acid, total cholesterol, and HDL at any of the time-points (Figure 6C–E). Consistent with our previous report, a significant reduction in TG levels was observed as early as the 4th week (118.8 mg/dL vs. 98.08 mg/dL) and remained reduced until the 12th week (126.7 mg/dL vs. 88.25 mg/dL) (Figure 6C–E).

**Figure 6.** *Cont*.

**Figure 6.** Body weight changes OA-control vs. OA-HA (**A**), or OA-control vs. OA-SNO and SNOHA (**B**). Metabolic profile of OA-control vs. OA-SNO rats (glucose, uric acid, total cholesterol, high-density lipoprotein (HDL), and triglyceride (TG)) at the 4th week (**C**), 8th week (**D**), and 12th week (**E**) after ACLT + MMx surgery. Values are expressed as the mean ± SEM, and two-way ANOVA and Tukey/Sidaks's multiple comparisons test were used to analyze the data. #/\* *p* < 0.05, ##/\*\* *p* < 0.01, ##/\*\*\* *p* < 0.001.
