Shi-style Steaming and Bathing Decoction on Knee Osteoarthritis by Decreasing Synovial Fibrosis and Angiogenesis

doi:10.21203/rs.3.rs-2224230/v1

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Research Article

Shi-style Steaming and Bathing Decoction on Knee Osteoarthritis by Decreasing Synovial Fibrosis and Angiogenesis

https://doi.org/10.21203/rs.3.rs-2224230/v1

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Aim of the study:

A discussion of the effect of Shi-style steaming and bathing decoction on synovial fibrosis and vascular hyperplasia.

Materials and methods

An anterior cruciate ligament transection (ACLT method) was used to replicate the knee osteoarthritis (KOA) model. After 4 weeks, and synovial tissues were taken for hematoxylin-eosin staining (HE) and Krenn score to verify whether the KOA model was successfully established. After the 4-week successful modeling, each group used different interventions for 4 weeks, while the sham-operated and KOA model groups were not intervened. At the end of the intervention, HE staining and Masson staining were used and Krenn scores and positive fiber areas were calculated.Femoral artery blood flows in the right lower limb was detected with a color Doppler ultrasound machine. The protein expression level of VEGFA was determined by enzyme-linked immunosorbent assay and immunohistochemical assay, and the quantitatively analyze the mRNA of VEGFA using Real-time PCR .

Results

The animals in Shi-style steaming and bathing decoction herbal bath treatment group generally performed well. HE staining, Krenn score and Masson staining indicated that Shi-style steaming and bathing decoction herbal baths reduced the number of synovial inflammatory cells, attenuated synovial fibrosis, decreased blood flow velocity, alleviated vascular resistance, and decreased the protein expression level of VEGFA in vivo and the mRNA expression level.

Conclusions

In the rabbit KOA model, Shi-style steaming and bathing decoction herbal bath at 40°C can alleviate synovial inflammation, fibrosis and revascularization, and improve local blood circulation of the knee joint. Therefore, it is feasible for the treatment of knee osteoarthritis.

Shi-style steaming and bathing decoction

Diclofenac Diethylamine Emulgel

Knee osteoarthritis

Synovial membrane

Rabbit

Anterior cruciate ligament

Knee osteoarthritis (KOA) is a chronic degenerative disease with multiple causative factors and complex pathogenesis. It has a high morbidity and disability rate, and consumes a large number of medical resources and social costs^[1]. The disease can cause synovial inflammation, joint tissue fibrosis, cartilage degeneration, bone flab formation, and other changes under the action of single or double stimulation of mechanical and chemical stimuli. The clinical symptoms can be seen as pain, swelling, and limited joint movement. Synovitis not only appears in the early stages of knee osteoarthritis^[2], but also accelerates the pathological course of the knee osteoarthritis. Synovitis can aggravate synovial angiogenesis and tissue fibrosis^[3], and synovial vascularization, in turn, drives the development of synovial inflammation and can lead directly to the development of synovitis^[4]. Tissue fibrosis in the pathological manifestation of knee osteoarthritis can lead to joint stiffness and pain^[5]. These are the main symptom and the main reason for KOA patients to seek medical attention^[6] .Therefore, controlling of inflammation and alleviating pain become the goal of treatment of the knee osteoarthritis. Evidence from relevant studies shows that^{[7, 8]} herbal fumigation therapy has a significant effect in eliminating inflammation and promoting synovial repair. The Shi’s herbal fumigation presciption is a centuries-old topical formula of the Shi’s Center of Orthopedics and Traumatology, and clinical studies have shown that the Shi’s herbal fumigation presciption has good efficacy in reducing swelling, alleviating pain and improving function in the treatment of the knee osteoarthritis^[9]. However, basic research has not yet been conducted. Based on the above, it was hypothesized that Shi-style steaming and bathing decoction could reduce the occurrence of synovial inflammation. In order to test this hypothesis, the present experimental study was conducted to observe the mechanism of action of Shi-style steaming and bathing decoction for treating knee osteoarthritis by decreasing synovial tissue fibrosis and vascular hyperplasia in the experimental rabbit KOA model induced by anterior cross transection at different temperatures to reduce the occurrence and decelerate the development of synovial inflammation.

Main reagents and instruments

Shi-style steaming and bathing decoction: 10g of Mahuang (Ephedra), 20 g of Guizhi (Gassia Twig), 10g of Xixin (Manchurian Wildginger), 20 g of Nanxing (Rhizoma Arisamatis), 20 g of Weilingxian (Chinese Clematis Root), 20 g of Baizhi (Dahurian Angelica Root), 20 g of Luhancao (Pyrola calliantha H. Andres), 10 g of Huajiao (Pricklyash Peel), 20 g of Dahuang (Rheum palmatum L.). The name already corresponds to the MPNS (http://mpns.kew.org). Chinese medicine is deployed from the Chinese medicine room of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine and supplied by Shanghai Kangqiao Chinese Medicine Beverage Co., Ltd. The beverage tablets used were tested by the quality management department of the company according to the Pharmacopoeia of the People's Republic of China 2020 edition a, "Shanghai Chinese medicine beverage tablet concoction specification" 2018 edition standard, and the results were all in accordance with the regulations; purified water (provided by the animal room); Diclofenac Diethylamine Emulgel (voltaren ointment, specification: 20 g, batch number VP2024 Ltd.); ELISA kit (Shanghai Enzyme Biotechnology Co., Ltd.); TRizol total RNA extraction kit (Takara); PCR detection kit (Takara); polymerase chain reaction primer synthesis (Shanghai Bioengineering Co., Ltd.); VEGF antibody (Invitrogen). Immunohistochemistry kit (Vector Laboratories); Gentamicin sulfate (Chongqing Pioneer Animal Pharmaceutical Co., Ltd.); Sodium pentobarbital powder (Sinopharm Chemical Reagent Co., Ltd.); Fresco D-37520 high-speed benchtop frozen centrifuge (Sorvall, USA); TP1020 tissue dehydrator, EG1150C biological tissue embedding machine, RM2165 type paraffin slicer, HI1210 type spreading machine, HI1220 type film drying machine (Leica, Germany); general optical microscope (Olympus, Japan), AG type color Doppler ultrasound instrument (Panasonic Video Cassette).

Establishment Of Koa Animal Model

In this experiment, 50 New Zealand rabbits weighing 2.0 ± 0.5 kg were purchased (Chedun Experimental Animal Breeding Farm, Songjiang District, Shanghai) and randomly divided into five major groups, including Shi-style steaming and bathing decoction herbal bath treatment group (16 rabbits), purified water bath group (16 rabbits), dichofenac diethylammon emulsion treatment group (6 rabbits), sham-operated group (6 rabbits), and KOA model group (6 rabbits), and the Shi-style steaming and bathing decoction herbal bath treatment group was further divided into Shi-style steaming and bathing decoction herbal bath 45 ℃ treatment subgroups, Shi-style steaming and bathing decoction herbal bath 40 ℃ treatment subgroups, Shi-style steaming and bathing decoction herbal bath 35 ℃ treatment subgroups, and the purified water bath group was divided into purified water bath 45 ℃ treatment subgroups, purified water bath 40 ℃ treatment subgroups, and purified water bath 35 ℃ treatment subgroups, with 5 rabbits in each group. All animals were housed at a temperature of (23 ± 2 ) °C, the humidity of (52 ± 3) %, and daytime light time of 12 h in individual cages. After 2 weeks of acclimatization feeding, the knee osteoarthritis model was constructed using classical anterior cruciate ligament resection (ACLT procedure)^[10]. Anesthesia was performed by intraperitoneal injection of 3% sodium pentobarbital solution at a dose of 1 mL/kg, and a medial parapatellar incision approach was taken to the medial knee joint with a transverse incision of approximately 1 cm in length, and the skin and subcutaneous tissue were incised to open the joint capsule. The right knee was fully flexed, and the patella was pushed outward to expose the joint cavity and expose the anterior cruciate ligament as much as possible. After confirmation, the anterior cruciate ligament was cut with a scalpel under direct vision. A positive “Lachman test” result indicated that the ACL had ruptured. The joint cavity was flushed with physiological saline and the joint capsule and incision were sutured. In the sham-operated group, only the skin and joint capsule were cut, but the ACL was not severed. Gentamicin sulfate (80,000 units) was administered intramuscularly to New Zealand rabbits from the day of surgery, once daily for 3 day to prevent wound infection. After the wounds being healed, the animals were driven around the room for 1 h a day, and a rabbit KOA model was obtained after 4 weeks. Postoperatively, antibiotics were administered continuously for 3 days to prevent infection. 5 rabbits were randomly executed after 4 week of surgical modeling and synovial tissue from the right knee joint was stained with HE and tested by Krenn score^[11]. The general condition and the results of HE staining of synovial tissues were combined to determine whether the model was successfully established.

Intervention Method

The intervention started 4 weeks after operation, and the daily dose of human drugs was converted to rabbit doses according to the “human-rabbit body surface area ratio”.^[12] In the Shi-style steaming and bathing decoction herbal bath group, the herbal solution was poured into a basin of water and the temperature was adjusted to 45°C,40°C and 35°C for 30 min each time a day for a total of 4 weeks (Fig. 1a). The purified water bath group was soaked in purified water, and the temperature in the water basin was adjusted to 45°C, 40°C, 35°C (Fig. 1b). The duration of operation was the same as that of the herbal bath group. We use an electronic thermometer to dynamically check the water temperature so that the temperature in the basin is maintained at an effective temperature of ± 2°C. The positive control group was given Diclofenac Diethylamine Emulgel latex topically applied to the knee joint at a dosage of 2 cm, once a day, with a 2 min of compression each time. Total intervention 4 weeks. The sham-operated group and the model group were not intervened.

Collection Of Tissue Samples

After 4 weeks of intervention, the animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1 kg/mL), and after the onset of anesthesia, blood was collected from the abdominal aorta in a sterile environment. Approximately 10 mL of blood was collected in a centrifuge tube and left for 6 h, followed by centrifugation at 3000 r/min for 10 min, and the supernatant was taken and stored in a refrigerator at -80°C. After complete exposure of the knee joint using a medial parapatellar incision, 1 mL of saline was taken with a 2 mL syringe and slowly injected into the joint cavity. The changes of the tissues around the joint cavity were closely observed during the injection, and the injection was stopped when it swelled. After repeatedly pumping back and injecting several times, the liquid was then withdrawn and injected into a labeled test tube, centrifuged at 3000 r/min for 10 min, and the supernatant was taken and sealed at -80°C for examination. The knee joint was flexed to fully expose the knee cavity, and the general condition of the synovial tissue was observed and recorded visually. The joint cavity was opened layer by layer, and synovial tissue was excised from the suprapatellar capsule, the deep infrapatellar capsule, the superior border of patellar cartilage, and the posterior medial crypt. Some were fixed in paraformaldehyde and some tissues were frozen in liquid nitrogen and subsequently transferred to -80°C refrigerator.

(He) And Krenn Score

Synovial tissues were fixed by immersion in paraformaldehyde for 24 h and then trimmed, dehydrated in an ethanol gradient, paraffin-embedded, sectioned to the thickness of 3 um, and stained with HE. A different field of view was selected for each section, and the pathological changes of synovial tissue were observed under light microscopy and scored using the Krenn scale^[11]. The Krenn scale was used for scoring. The scoring criteria were as follows: observation of the degree of hyperplasia of the synovial lining layer, no hyperplasia/enlargement of the synovial lining layer (0 score), slight increase of 2–3 layers, very rare giant cells (1 point), severe increase to 4–5 layers with a few giant cells or lymphocytes (2 points), severe increase (> 5 layers), ulceration of the lining, many giant cells and lymphocytes (3 points); degree of synovial stromal activation; no activation of the synovial stroma, normal cell structure (0 point), mildly increased synovial stroma (hypocellularity with mild edema, mild fibrosis, few fibroblasts, no giant cells) (1 point), moderate activation of synovial stroma (moderate cellularity with moderate density of fibroblasts, endothelial cells, giant cells) (2 points), highly activated synovial stroma (hypercellularity, dense distribution of fibroblasts and endothelial cells, abundant giant cells) (3 points); inflammatory cell infiltration degree, no inflammatory infiltration (0 point), mild inflammatory infiltration (distribution of extensive monocytes and small lymphocytes/plasma cells aggregated around the vessels) (1 point), moderate inflammatory infiltration (perivascular/superficial lymphoid aggregates observed, no occurrence of small lymphoid follicles in the center) (2 points), and severe inflammatory infiltration (formation of lymphoid follicles in the center of birth or subsynovial confluent lymphoid infiltration) (3 points). The total score is calculated and can be divided into no synovitis (total score 0–1), mild synovitis (total score 2–4), and severe synovitis (total score 5–9).

Masson

Paraffin sections of synovial tissues were examined microscopically after Masson staining according to the reagent instructions, and each section was randomly photographed in 5 different fields of view (×200), and the percentage of positive fibrous structures in synovial tissues was counted and analyzed according to the images through Image J software, and the average value was taken. The proportion of blue positive fibers in synovial tissue to the synovial area of the corresponding field was calculated and analyzed by Image J software.

Doppler Ultrasound Detection Of Changes In Right Femoral Artery Blood Flow

We use Doppler ultrasonography and keep the instrument parameters constant during the examination. After palpating the pulsation of the right femoral artery by hand, the probe is selected and placed at the pulsation of the femoral artery for detection, noting that the probe angle is always < 60 °. After optimizing the image, the internal diameter of the vessel is measured manually and the corresponding hemodynamic parameters, such as peak systolic flow velocity (PSV), end-diastolic flow velocity (EDV), vascular resistance index (RI), an internal diameter of the femoral artery, are obtained (Fig. 2). All data are saved in the computer for further analysis.

Enzyme-linked Immunosorbent Assay (Elisa)

ELISA was performed to detect the protein content of VEGF in serum and joint fluid. The serum and joint fluid samples of each group were rewarmed at room temperature 1h in advance, and the concentration of VEGFA in serum and joint fluid were detected by ELISA kit. Three replicate apertures were set up for each sample.

Real-time Polymerase Chain Reaction (Real-time Pcr)

Real-Time PCR was performed to detect the expression of VEGFA mRNA in synovial tissue with the following primer sequences: internal reference

GAPDH (Forward 5′-AGAGCACCAGAGGAGGACGA-3′;

Reverse 5′-TGGGATGGAAACTGTGAAGAGG-3′);

VEGFA (Forward 5′- AACGAACGTACTTGCAGATGT-3′;

Reverse 5′-GCTCACGCAGTCTCCTCTTC-3′). Total RNA was extracted from synovial tissues by Trizol reagent. the concentration and purity of extracted RNA were detected using UV absorption, reverse transcription was performed using a first-strand cDNA synthesis kit with gDNA, gene expression was detected using a qPCR kit, and quantitative PCR results were analyzed using Applied Biosystems 7500 Real-Time PCR system software and expressed as ploidy changes in cDNA levels, and final analysis was performed using the 2^−ΔΔCTmethod^[13].

Immunohistochemistry (Ihc)

The slices were baked at 60°C for 2 h before the experiment, dewaxed to water by conventional methods, heated in a sodium citrate buffer water bath for 15 min at 95°C for antigen repair, and incubated with endogenous peroxidase blocking reagent for 10 min at room temperature. Add protein blocking solution dropwise and incubate VEGF antibody (diluted 1:50) for 20 min at room temperature overnight at 4°C. Sections incubated in phosphate buffer saline (PBS) without antibodies can be used as negative controls. After incubation with biotinylated secondary antibodies and affinity-biotin complex reagent containing horseradish peroxidase for 30 min, sections are visualized with DAB development and hematoxylin re-staining. Image-Pro Plus 6.0 system (IPP) image analysis system is used for quantitative analysis. The results were observed under light microscopy, and 5 fields of view were selected for each section; positives were brown or tan and granular. Image analysis: 5 high magnification fields (×200) were randomly selected and the average optical density value (AOD) of each field was calculated and finally averaged. AOD = IOD/Area.

Statistical analysis

The data measured were expressed as mean ± standard deviation (‾x ± s) using SPSS 24.0 statistical software, and comparisons between multiple groups were analyzed by analyzing one-way ANOVA (LSD method if the variance was the same; Dunnett’s T3 method if the variance was not the same). The test level was α = 0.05, and P < 0.05 was used as the significance level. Plots were performed using Graphpad Prism 8.0 software.

The general conditons of animals

Throughout the experiment, there were no obvious abnormalities in the animals of each group. One day after modeling, the sham-operated group showed no obvious abnormal changes in feeding, defecation, mental status, fur, and both knee joints moved freely. Animals in the model group, Shi-style steaming and bathing decoction herbal bath treatment group, purified water bath treatment group, and Diclofenac Diethylamine Emulgel emulsion treatment group showed anorexia, poor mental status, obvious knee joint redness, and swelling, and poor joint movement; 3 days after the operation, mental diet returned to normal, knee joint redness and swelling were still obvious; the wound of the affected limb healed 5–7 days after an operation, no infection in the wound during healing, and knee joint swelling returned to normal; At 4 weeks after the operation, the affected knee was swollen and deformed compared to the healthy side, with varying degrees of claudication and average knee mobility. After 4 weeks of intervention, the treatment and control groups had moderate arthrogryposis of the knee, average joint mobility, and improved claudication. The 40°C subgroups performed better. Animals in the model group had significantly enlarged knees, severe joint deformities, significantly restricted joint mobility, and the presence of large amounts of joint effusion (Fig. 3).

Test Index Results

Gross morphological observation

After the 4-week intervention, the synovial membrane of the affected knee in the sham-operated group was uniformly distributed, without thickening, with a softer texture, a light yellow color, and a shiny hue. In the model group, the synovial membrane was dark red, adherent to the surrounding tissues, tougher in texture, darker in color, and more capillary hyperplasia can be seen. The synovial tissue in the Shi-style steaming and bathing decoction bath treatment group was slightly thickened, with a dark yellow color and slightly shiny. The general observation of synovial tissue in the 40°C treatment subgroups was better than that in the 35°C and 45°C treatment subgroups. In the purified water bath treatment group, the synovial membrane of the affected knee was thickened significantly, with localized hyperplastic adhesions, slightly tough, dark yellow, and decreased luster, in which the purified water bath 40°C treatment subgroups performed better than the water bath 35°C treatment subgroup and the water bath 45°C treatment subgroups. The synovial tissue in the Diclofenac Diethylamine Emulgel treatment group was significantly thickened, with a light-dark yellow color and reduced coloration compared to the sham group.

Synovial He Staining

To determine whether the Shi-style steaming and bathing decoction could reduce synovitis through anti-inflammatory effects, we performed HE staining and had two non-participating staff members Krenn score the sections. The 4-week sham-operated group: flattened synovial cells were seen in an orderly monolayer with low cell numbers, no inflammatory cell infiltration or interstitial edema, and no significant vascular hyperplasia (Fig. 4)., and the 8-week results were generally consistent with the 4-week results (Fig. 5). After 4 weeks of modeling, the synovial tissue in the surgical modeling group was more hyperplastic, with an increased number of synovial cells, disorganized arrangement, local inflammatory cell infiltration between cells, and increased fibrosis under the synovial tissue (Fig. 4). The Krenn score was 4 in all groups except the sham-operated group, indicating that surgical modeling in this experiment could cause moderate synovitis and successful modeling (Table. 1). histopathological changes in the KOA model group after 8 weeks remained the same as those at 4 weeks, but the degree of inflammation increased (Fig. 5). Synovial cell proliferation, hypertrophy, inflammatory infiltration profile, and cellular arrangement order were superior to those of the KOA model group after intervention with Shi-style steaming and bathing decoction herbal bath, purified water bath, and Diclofenac Diethylamine Emulgel emulsion, with the former group performing better at a temperature of 40°C (Fig. 5.). The overall Krenn score after 8 weeks was lower than the Krenn score before 4 weeks (Fig. 6).

Masson Staining Of A Synovial Membrane

To test the anti-fibrotic effect of the Shi-style steaming and bathing decoction in vivo, we performed Masson staining for the synovial tissue of experimental rabbits, after which the staining showed red coloration for muscle fibers and cytoplasm and blue coloration for collagen fibers, and the shades of coloration indicate the number of collagen fibers. A small number of light blue fibers can be seen in the synovial membrane of the sham-operated group and the herbal bath group, and the proliferation of synovial cells was not obvious; the synovial membrane of the KOA model group and the purified water bath treatment group was heavily fibrotic, with blue In the KOA model group and the purified water bath group, the synovial membrane was heavily fibrotic with deep blue staining and different degrees of synovial cell hyperplasia; the synovial membrane in the Diclofenac Diethylamine Emulgel treatment group was significantly less fibrotic with mild to moderate fibrosis (Fig. 7). Semi-quantitative results of the percentage of collagen area positive fibers in Masson stained sections showed that compared with the sham-operated group, the fiber content in the KOA model group, the Shi-style steaming and bathing decoction herbal bath treatment group, the purified water bath treatment group, and the Diclofenac Diethylamine Emulgel treatment group was significantly higher; compared with the KOA model group, the use of the herbal bath, purified water bath, and Diclofenac Diethylamine Emulgel inhibited and decreased the fibrosis of synovial membrane, and the reduction was more significant with the use of Shi-style steaming and bathing decoction herbal bath at 40 ℃ (Fig. 7j).

Color Doppler Examination

To study the effect of Shi-style steaming and bathing decoction on femoral artery blood flow in the lower extremities of New Zealand rabbits, we used color Doppler ultrasound for measurements. In this experiment, the peak systolic flow velocity, peak diastolic flow velocity, and femoral artery internal diameter were higher in the Shi-style steaming and bathing decoction herbal bath treatment group, the purified water bath treatment group, and the Diclofenac Diethylamine Emulgel treatment group than in the KOA model group and the sham-operated group, but the vascular resistance was less than that of the KOA model group (Table. 2).

Detection Of Protein And Gene Expression Of Vegfa

To investigate whether Shi-style steaming and bathing decoction can reduce angiogenesis, we used ELISA to detect the protein expression of VEGFA in serum and joint fluid of each group. The data showed that, compared with the sham-operated group, the VEGFA content was significantly increased in the KOA model group, the Shi-style steaming and bathing decoction herbal bath treatment group, the purified water bath treatment group, and the Diclofenac Diethylamine Emulgel treatment group. Compared with the model group, the use of Shi-style steaming and bathing decoction herbal bath, purified water bath, and Diclofenac Diethylamine Emulgel could inhibit the increase of VEGFA, and the decrease in Shi-style steaming and bathing decoction herbal bath group was more significant; the VEGFA content also showed different degrees of decrease after different temperature interventions. The VEGFA content also decreased to different degrees after different temperature interventions, but the most obvious VEGFA content was found in the Shi-style steaming and bathing decoction herbal bath at 40°C (Fig. 8). The use of Real-Time PCR further confirmed that Shi-style steaming and bathing decoction herbal bath at 40°C reduced angiogenesis (Fig. 9).

Immunohistochemistry Of Vegf

The positive side of VEGF staining was mainly the presence of brownish-yellow granules in the cytoplasm and/or cytosol. The positive cells were expressed in small amounts in the sham-operated group, and the positive expression rate was elevated in the KOA model group, the Shi-style steaming and bathing decoction herbal bath treatment group, the purified water bath treatment group, and the Diclofenac Diethylamine Emulgel treatment group, compared with the sham-operated group, and the mean optical density value was lowest in the Shi-style steaming and bathing decoction bath treatment group, (Fig. 10).

KOA is a common and disabling form of arthritis, the prevalence and incidence of which are increasing every year.^[14] Currently, oral or topical NSAIDs, corticosteroid injections into the joint cavity, hyaluronic acid compounds, platelet plasma, opioids, non-opioids, and antidepressants have failed to slow down the progression of KOA^[14], so finding new drugs to slow down the progression of KOA remains the focus of our research.

Related evidence ^{[15, 16]}suggests that topical bath therapy can relieve pain and stiffness and improve the quality of life of patients with knee osteoarthritis. We conducted a literature search and found that studies on the use of traditional Chinese herbal bath for the treatment of the knee osteoarthritis and the exploration of appropriate bath temperatures for traditional Chinese herbal bath^{[17, 18]}. Based on this temperature, we re-set three temperature gradients for verification.

In the present study, the ACLT method was chosen to model KOA, a method that leads to severe inflammation after 4 weeks, with cartilage degeneration, and other early pathological changes characteristic of KOA^[19]. Therefore, that time point was chosen for intervention using drugs, and we expected that Shi-style steaming and bathing decoction herbal bath treatment group would show anti-inflammatory properties and alleviate angiogenesis, which in turn would reduce pain and delay the progression of KOA in patients

The anti-inflammatory properties of Shi-style steaming and bathing decoction were investigated by calculating the synovitis score of HE sections. Compared with the control group, synovial cell hypertrophy, hyperplasia, stromal edema and inflammatory cell infiltration were obvious in the synovial HE staining of the 4-week and 8-week groups. The inflammatory cells were reduced and the severity of hypertrophy, hyperplasia, and edema was significantly lower in Shi-style steaming and bathing decoction herbal bath treatment group compared with the purified water bath treatment group and the Diclofenac Diethylamine Emulgel treatment group.

In patients with KOA, synovial cells secrete vascular endothelial growth factor, which increases local angiogenesis, which in turn transports inflammatory factors and increases vascular permeability, thereby perpetuating the inflammatory response^[20]. VEGF is an important vascular growth factor secreted by endothelial cells, which stimulates angiogenesis, increases vascular permeability, and dilates blood vessels^[21]. Angiogenesis is the production of new capillaries in the pre-existing vascular system^[22]. Increased synovial angiogenesis and elevated expression of VEGF promote synovial inflammation^[21]. Synovial inflammatory pathology, including synovial tissue thickening, inflammatory cell infiltration, and fibrosis^[23]. Since HE sections of synovial tissue showed a reduction in angiogenesis in the Shi-style steaming and bathing decoction herbal bath treatment group, we used color Doppler ultrasound to detect blood flow in the femoral artery of the right lower extremity, and further applied ELISA and IHC to detect the protein expression of VEGF and Real-Time PCR to detect gene levels of VEGF to determine the effect of reducing synovial angiogenesis and slowing down the progression of KOA. Previous evidence suggests that VEGF expression levels in the synovial fluid of patients with knee osteoarthritis are tenfold higher than in healthy normal groups, with higher positive expression in cartilage and synovial tissues and a positive correlation with the severity of KOA imaging^[22]. VEGF was expressed in the serum, synovial fluid, and synovial fluid of KOA patients^[24]. synovial fluid^{[25, 26]} subchondral bone^[27], and meniscus^[28] VEGF was expressed in serum, synovial fluid, subchondral bone, and meniscus of KOA patients. Injecting VEGF into normal knee joints can promote synovial proliferation^[29] and conversely, injection of anti-VEGF can reduce the occurrence of synovial inflammation.^[30]. This is consistent with our experimental results. In addition, there are also related studies reporting^[31] that anti-VEGF can reduce pain in patients with knee osteoarthritis, and blocking the expression of VEGF can inhibit the excitation of sensory neurons and reduce the sensitivity of pain^[32].

Synovial fibrosis is one of the causes of pain and a feature of synovitis, often occurring during the progression of KOA^{[33, 34]}. Fibrosis of the synovium is accompanied by angiogenesis and is highly correlated with VEGF^[35]. The blood supply to the synovial membrane accelerates the development of synovial fibrosis and VEGF reduces the anti-fibrotic effect^{[36, 37]}. The severity of synovial fibrosis after ACLT was assessed by examining the synovial membrane of the knee using Masson trichrome staining. The collagen volume fraction in Masson trichrome stained sections showed that the collagen content increased and fibrosis became severe after 8 weeks of modeling compared to the Sham-operated group. Intervention with the Shi-style steaming and bathing decoction herbal bath reduced collagen deposition and decreased fibrosis in the synovium.

The limitations of the study in this experiment are as follows

firstly, this experiment was conducted on a New Zealand Great White rabbit KOA model with severed anterior cruciate ligament, and the pathological changes of the knee joint in rabbits are different from those of human KOA, which need to be further studied in human clinical trials. Secondly, the experiment did not involve illustrating the relationship between pain and fibrosis. Thirdly, many inflammatory factors can directly or indirectly regulate the expression of VEGF. Fourth, this experiment was conducted only on serum, synovial fluid, and synovial tissues, but in fact, VEGF is expressed in cartilage, subchondral bone, and meniscus of KOA patients, and we plan to expand our research in this area in the future to better understand the disease. In addition, this experiment used a Chinese herbal compound to intervene in the treatment, but we were unable to clarify the effective components of its action, and failed to experiment with the perspective of pharmacokinetics and pharmacokinetics, which is also the future research direction of Chinese herbal compound.

In summary, our study demonstrated that the Shi-style steaming and bathing decoction herbal bath at 40°C reduced the infiltration of inflammatory cells and inhibited the vascular proliferation of the synovial membrane, which could promote faster blood flow to the lower extremities, reduce vascular resistance and fibrosis of the synovial membrane, and effectively alleviate the inflammation. These results suggest that Shi-style steaming and bathing decoction herbal bath is a potentially effective treatment for patients with knee osteoarthritis, but further in-depth studies are needed to fully clarify the mechanism of action of Shi-style steaming and bathing decoction for knee osteoarthritis.

OA	Osteoarthritis
KOA	Knee osteoarthritis
ACLT	Rupture of the anterior cruciate ligament
VEGF	Vascular endothelial growth factor
VEGF-A	Vascular endothelial growth factor-A
ELISA	Enzyme-linked immunosorbent assay
Real-Time PCR	Real-Time Polymerase Chain Reaction
IHC	Immunohistochemistry
PSV	Peak Systolic Velocity
EDV	Early Retrograde Diastolic
RI	Resistance Index
AOD	Average Optical Density

Ethical approval and consent to participate

All experimental protocols were conducted under the guidelines developed by the Animal Experimentation Center of Shanghai University of Traditional Chinese Medicine and approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. (Number: PZSHUTCM201231003).

Agree to publish

CrediT authorship contribution statement

Shengyue Wen, Biao Tian, and Bo Chen:jointly completed the experimental design. Shengyue Wen and Biao Tian:performed the animal rearing, intervention, and sampling. Shengyue Wen: performed the sample testing and data processing. Shengyue Wen and Bo Chen: conceptually designed the study and prepared the manuscript. All authors contributed to the article and approved the submitted version.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled “Shi-style Steaming and Bathing Decoction Herbal Bath Treatment on Knee Osteoarthritis by Decreasing Synovial Fibrosis and Angiogenesis”. No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication

Data availability

Data will be made available on request.

Acknowledgments

This project was funded by the National Natural Science Foundation of China (81774340), the Shanghai Science and Technology Innovation Action Plan Medical Innovation Research Special Project (21Y11921600), and the Shanghai Clinical Medical Center for Chronic Musculoskeletal Diseases (20MC1920600).

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Table 1

Krenn score of synovial tissue after 4 weeks(‾x ± SD)
Group	N	Lining Layer Hyperplasia	Activation Of Resident Cells	Inflammatory Infiltrate	Krenn Score
Sham	1	0	0	0	0
Model	1	1.5	1	1.5	4
DDE	1	1.5	1	1.5	4
HB	1	1.5	1	1.5	4
WB	1	1.5	1	1.5	4
Note: Sham: Sham-operated group;Model:KOA model group; DDE:Dichofenac Diethylammon emulsion treatment group;HB: Shi-style steaming and bathing decoction herbal bath treatment group; WB: purified water bath treatment group.

Table 2

Right lower extremity femoral artery hemodynamic index measurements(‾x ± SD)
Group/subgroups	N	PSV(cm/s)	EDV(cm/s)	RI	Internal Diameter(mm)
HB 45 ℃	5	49.39 ± 2.32^*#	17.01 ± 1.64^*▽	0.64 ± 0.05^*	1.16 ± 0.02^*#
HB 40 ℃	5	52.80 ± 1.82^*#	18.74 ± 2.27^*	0.60 ± 0.02^*	1.18 ± 0.03^*#
HB 35 ℃	5	48.96 ± 0.98^*△	16.62 ± 2.17^*▽	0.70 ± 0.05^*△▽	1.14 ± 0.02^*△
WB 45 ℃	5	49.71 ± 1.83^*△	16.01 ± 2.55^*△▽	0.68 ± 0.02^*△▽	1.15 ± 0.01^*
WB 40 ℃	5	50.29 ± 1.48^*	16.47 ± 1.45^*▽	0.66 ± 0.03^*△▽	1.16 ± 0.02^*#
WB 35℃	5	48.12 ± 3.42^*△▽	15.21 ± 1.52^△▽	0.71 ± 0.04^*△▽	1.14 ± 0.02^*△▽
DDE	5	51.71 ± 2.58^*#	19.99 ± 1.69^*#	0.60 ± 0.07^*#	1.17 ± 0.02^*#
Sham	5	47.76 ± 2.40^*△▽	17.09 ± 1.33^*▽	0.66 ± 0.04^*	1.12 ± 0.02^*▽
Model	5	38.19 ± 2.46^#△▽	12.96 ± 1.81^#△▽	0.77 ± 0.05^#△▽	1.03 ± 0.04#△▽
Note: PSV,Peak Systolic Velocity;EDV,Early Retrograde Diastolic;RI,Resistance Index. HB 45℃: Shi-style steaming and bathing decoction herbal bath 45 ℃ treatment subgroups; HB 40 ℃: Shi-style steaming and bathing decoction herbal bath 40 ℃ treatment subgroups; HB 35 ℃: Shi-style steaming and bathing decoction herbal bath 35 ℃ treatment subgroups; WB45℃:purified water bath 45 ℃ treatment subgroups; WB 40 ℃:purified water bath 40 ℃ treatment subgroups; WB 35 ℃: purified water bath 35℃ treatment subgroups; DDE:Dichofenac Diethylammon emulsion treatment group; Sham: Sham-operated group; Model:KOA model group;., *P < 0.05; compared with the sham-operated group, #P < 0.05; compared with the herbal bath 40°C subgroups, △P < 0.05; compared with the Diclofenac Diethylamine Emulgel group, ▽P < 0.05.

No competing interests reported.

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Shi-style Steaming and Bathing Decoction on Knee Osteoarthritis by Decreasing Synovial Fibrosis and Angiogenesis

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Abstract

Aim of the study:

Materials and methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Main reagents and instruments

Establishment Of Koa Animal Model

Intervention Method

Collection Of Tissue Samples

(He) And Krenn Score

Masson

Doppler Ultrasound Detection Of Changes In Right Femoral Artery Blood Flow

Enzyme-linked Immunosorbent Assay (Elisa)

Real-time Polymerase Chain Reaction (Real-time Pcr)

Immunohistochemistry (Ihc)

Statistical analysis

Results

The general conditons of animals

Test Index Results

Gross morphological observation

Synovial He Staining

Masson Staining Of A Synovial Membrane

Color Doppler Examination

Detection Of Protein And Gene Expression Of Vegfa

Immunohistochemistry Of Vegf

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

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