HE Yueyi (何岳義), MAO Zhen (毛珍)

1 Meishan Traditional Chinese Medicine Hospital, Sichuan Province, Sichuan 620000, China

2 People’s Hospital of Caidian District, Wuhan City, Hubei Province, Wuhan 430100, China

Abstract

Keywords: Tuina; Massage; Manual Manipulation; Moxibustion Therapy; Heat Sensitive Moxibustion; Osteoarthritis, Knee;ADAMTS4 Protein; Matrix Metalloproteinase 3

Osteoarthritis (OA) is a progressive joint disease featured by cartilage degeneration[1], mainly affecting the load-carrying joints such as hips and knees[2], and has become the most common arthritis[3].Asymptomatic OA has a higher prevalence rate, and about 0.25 billion people are suffering from OA worldwide[4]. The morbidity rate of knee osteoarthritis(KOA) has increased significantly over the last decades.The prevalence of KOA ascended by 32.7% between 2005 and 2015, making OA one of the leading causes of disability[5]. The risk factors for KOA include age, knee injuries, increased body mass, joint discomfort, and high mechanical stress[6-7]. It is already known that the primary characteristics of KOA are the altered structure of cartilages and subchondral bones and lesions involving Hoffa’s fat pad, synovial membranes,ligaments, and muscles. However, its exact etiology and pathogenesis are still veiled[8].

In the early stage of KOA, the cartilage matrix alters under the action of degrading enzymes. C-telopeptide of type Ⅰ collagen (CTX-Ⅰ) is a specific indicator that can reflect the degradation of type Ⅰ collagen.Tartrate-resistant acid phosphatase 5b (TRACP-5b) can accelerate the degradation of calcium phosphate mineralized substrates. The two elements mainly reside in osteoclasts and can effectively evaluate bone absorption and osteoclast function[9-10]. A disintegrin and metalloproteinase with thrombospondin 4(ADAMTS-4) is a hydrolytic enzyme in the extracellular matrix of articular cartilage. So is matrix metalloproteinase 3 (MMP-3), which participates in matrix degradation and OA development. They are two significant markers evaluating cartilage degradation intensity[11-12]. Research has found a notably higher ADAMTS-4 level in early-stage OA than in the advanced stage and healthy controls; comparatively, a markedly higher MMP-3 level has been found in the advanced-stage OA than in the early stage and healthy controls[13].

Knee-balancing manipulation is a kind of Tuina(Chinese therapeutic massage) therapy targeting the imbalance between tendons and bones (structural malposition) to restore balance by loosening up tendons and adjusting bones. This manipulation takes care of the general structure. It aims to regain the balance of the general structure and physical force line through three steps, i.e., loosening up tendons,adjusting bones, and restoring balance, starting from the pelvic bone downward[14]. A recent study revealed that heat-sensitive moxibustion could effectively mitigate the clinical symptoms, down-regulate inflammatory factor levels, and lower the adverse reaction rate in treating KOA patients. Its efficacy was more significant compared with the ordinary moxibustion treatment[15]. Our previous work has also demonstrated that electroacupuncture at the “painsensitive point” plus heat-sensitive moxibustion can improve the symptoms and blood lipid levels in KOA patients[16]. Besides, we have also proved that KOA due to Yang deficiency and cold coagulation is positively correlated to CTX-Ⅰ, TRACP, ADAMTS-4, and MMP-3 levels (P＜0.05)[17]. Therefore, we conducted this indepth research focusing on the impact of kneebalancing manipulation plus heat-sensitive moxibustion on CTX-Ⅰ, TRACP-5b, ADAMTS-4, and MMP-3 to discuss the preventive and treatment efficacy and mechanism of this treatment method for KOA. The purpose is also to provide scientific evidence to back up the clinical application of this approach in preventing and treating KOA and related medical conditions.

1 Clinical Materials

1.1 Diagnostic criteria

The study referred to the diagnostic criteria for KOA reformulated by the American College of Rheumatology[18]. (1) Suffered from knee joint pain most of the time during the recent one month; (2) X-ray images revealed osteophytes on the joint brim; (3)synovial fluid examination results were consistent with OA; (4) age over 40 years old; (5) morning stiffness≤30 min; (6) bone fricative during joint movements.KOA could be diagnosed when the patient met items (1)and (2) or (1) (4) (5) and (6).

1.2 Inclusion criteria

Met the diagnostic criteria; aged between 40 and 75 years; agreed to participate in the trial, and signed the informed consent form.

1.3 Exclusion criteria

Those with complications that affected the knee joint;pregnant or lying-in women and those with endocrine disorders; those with cardiac, liver, kidney, or brain injuries or severe congenital diseases; those with contraindications for moxibustion.

1.4 Rejection or dropout criteria

Those with a poor compliance or failure to complete required treatment; those with incomplete data that hindered result analysis; those who showed severe adverse reactions or complications; those who asked to quit on their own; those who used other medications or received other treatments without permission.

1.5 Statistical methods

The SPSS version 22.0 statistical analysis software was applied. The measurements were expressed as mean ± standard deviation (±s). Those distributed normally and passing the homogeneity of variance test were compared using the analysis of variance in multiple-group comparisons and the Student-Newman-Keuls test for between-group comparisons. Those not meeting normal distribution or homogeneity of variance were checked using the non-parametric test.The enumeration and ranked data were presented as case numbers or percentages and analyzed using the Chi-square test. Statistical significance was accepted whenP＜0.05.

1.6 Sample size

We determined the sample size according to the pretest results using the PASS 15.0 multi-group comparison method. When we set the mean total score of the Western Ontario and McMaster Universities arthritis index (WOMAC) as 19, 18, and 14, respectively,in the three groups, their standard deviation would be 3.2, 6.3, and 2.7. A two-tailed test was demanded withα=0.05 and the power of test =90%. We then determined the sample size asn=111 using the one-way analysis of varianceF-test in the PASS software. Finally,when assumed a lost-to-visit or refusal-to-visit rate of 20% and decided to recruit at least 134 participants.

1.7 Safety rating

Any adverse events and management were recorded during the treatment, including itching, pain, blisters,burns, infection, and allergies. The adverse events were graded into three levels. Mild: occasional mild symptoms during the treatment lasting less than 0.5 h;moderate: mild symptoms right after or a while after treatment lasting less than 2 h; severe: serious symptoms right after moxibustion treatment lasting over 2 h and intolerable.

1.8 General data

The participants were all KOA patients recruited from the Rehabilitation Medicine Department of Meishan Traditional Chinese Medicine Hospital between March 2019 and March 2021. The 134 subjects were coded.We used the SPSS version 21.0 software to generate random seeds and then sequenced the obtained random numbers and divided them into three groups, a knee-balancing group (44 cases), a heat-sensitive moxibustion group (44 cases), and a joint intervention group (46 cases). The randomized allocations were hidden in sealed opaque envelopes waiting for the eligible participants to open and receive the corresponding treatment. During the intervention, one case in the knee-balancing group dropped out; another two dropout cases were from the joint intervention group. The three groups had no significant differences in the general data (P＞0.05), suggesting comparability.The details are shown in Table 1.

The Ethics Committee of Meishan Traditional Chinese Medicine Hospital approved this trial (Approval No.CDRMYY-20191102001), and the patients signed the informed consent form.

Table 1. Comparison of the general data between the two groups

2 Treatment Methods

2.1 Knee-balancing group

The first step, loosen up tendons. The patient took a supine position. The physician first Tui-Pushed and Nian-Twisted around the knee to relax the skin, then relaxed fasciae, followed by Rou-Kneading, Na-Grasping,Tanbo-Plucking, Tui-Pushing, and Dian-Digital An-Pressing manipulations to relax the low back,buttock, and leg muscles. The second step, adjust bones.The patient took a supine position. The “question mark”and “inverse question mark” methods were used to correct genu varum or genu valgum, the “drawer”method was adopted to repair the anterior or posterior displacement of the tibia, and the circularly Tui-Pushing manipulation was applied to correct the position of the patella. The third step, restore balance and rebuild the force line of the joint. The patient took a prone position with the affected-side lower limb straight. Following adjusting bones, the physician repeated Rou-Kneading,Na-Grasping, Tanbo-Plucking, Tui-Pushing, and Dian-Digital An-Pressing manipulations to relax the low back, buttock, and leg muscles. Then, the patient took a supine position. The physician Tui-Pushed and An-Pressed the tendons around the knee joint. The treatment took 20 min each and was conducted once a day, 5 times a week for a total of four weeks.

2.2 Heat-sensitive moxibustion group

The patient took a supine position. The physician first performed mild moxibustion through Yinlingquan (SP9),Yanglingquan (GB34), Liangqiu (ST34), and Xuehai (SP10)on the side of the affected knee. Then, when the patient felt the moxibustion heat penetration (the moxibustion heat transmits from the body surface to the deep layers), expansion (the heat spreads around),or transmission (the heat transmits from the treated spot toward another direction), we knew that the point heat-sensitization was triggered. The triggered point was considered a heat-sensitive point. Finally, four heat-sensitive points were selected and recorded. Each point received moxibustion for 20 min, once a day,5 times a week for a total of four weeks.

2.3 Joint intervention group

Patients in the joint intervention group received the same knee-balancing manipulation and heat-sensitive moxibustion once a day, 5 times a week for four weeks.

3 Therapeutic Efficacy Observation

3.1 Observation items

Before and after treatment, as well as at the 2-week and 6-week follow-ups, the three groups were observed using the visual analog scale (VAS) for knee joint pain and WOMAC (including pain, stiffness, and daily activities)[19].

At the same time points, 5 mL fasting venous blood was collected to run the enzyme-linked immunosorbent assay for detecting the contents of serum CTX-Ⅰ,TRACP-5b, ADAMTS-4, and MMP-3.

3.2 Criteria for therapeutic efficacy

We referred to the efficacy criteria for KOA in theGuiding Principles for Clinical Study of New Chinese Medicines[20]for assessing the efficacy after treatment and at the 2-week and 6-week follow-ups. WOMAC score reduction rate = (WOMAC score before treatment - WOMAC score after treatment) ÷ WOMAC score before treatment × 100%.

Recovered: The primary knee symptoms were gone,the joint motion became normal, and the WOMAC score reduction rate was ≥80%.

Markedly effective: The primary knee symptoms showed notable improvements, the joint motion was almost normal, and the WOMAC score reduction rate was ≥50% but ＜80%.

Effective: The primary knee symptoms and joint motion showed improvements, and the WOMAC score reduction rate was ≥25% but ＜50%.

Invalid: There showed no improvements but even aggravation in the primary symptoms and joint motion,and the WOMAC score reduction rate was ＜25%.

3.3 Results

3.3.1 Comparison of the clinical efficacy

In the joint intervention group, the total effective rate was 93.2%, 90.1%, and 86.4% after treatment and at the 2-week and 6-week follow-ups, respectively; higher than that in the knee-balancing and heat-sensitive moxibustion groups, and the between-group differences were statistically significant (P＜0.05). The data are detailed in Table 2.

Table 2. Comparison of the clinical efficacy at different time points (case)

3.3.2 Comparison of the VAS score

Compared with the baseline, the VAS score showed significant decreases at the three time points in each group (P＜0.05). The results suggest that the kneebalancing manipulation and heat-sensitive moxibustion can produce stable, long-lasting efficacy in reducing the pain in KOA patients, either used separately or combined. The three groups had no significant differences in the VAS score before treatment (P＞0.05).The VAS score was notably lower in the joint intervention group than in the knee-balancing and heat-sensitive moxibustion groups at the three time points after treatment (P＜0.001). The results indicate that the pain reduction effect is most significant in the joint intervention group (Table 3).

3.3.3 Comparison of the WOMAC score

Compared with the baseline, the WOMAC component and total scores decreased at the three time points in each group, and the intra-group differences were statistically significant (P＜0.05). The results suggest that either used alone or together, the knee-balancing manipulation and heat-sensitive moxibustion can effectively ease the pain and stiffness and improve joint function in KOA patients. The three groups had no significant differences in the WOMAC component or total scores before the treatment(P＞0.05). At the three time points, the WOMAC component and total scores were significantly lower in the joint intervention group than in the knee-balancing and heat-sensitive moxibustion groups (P＜0.001). It is demonstrated that the knee joint function is the best in the joint intervention group after treatment. The details are shown in Table 4-Table 7.

3.3.4 Comparison of the expression levels of serum CTX-Ⅰ, TRACP-5b, ADAMTS-4, and MMP-3

Compared with the baseline, the expression levels of CTX-Ⅰ, TRACP-5b, ADAMTS-4, and MMP-3 dropped at the three time points in each group (P＜0.05). The results suggest that the knee-balancing manipulation and heat-sensitive moxibustion can produce stable,long-lasting effects in down-regulate the expression levels of CTX-Ⅰ, TRACP-5b, ADAMTS-4, and MMP-3 either used alone or together. The three groups had no significant differences in the expression levels of CTX-Ⅰ,TRACP-5b, ADAMTS-4, or MMP-3 before treatment(P＞0.05). The expression levels of CTX-Ⅰ, TRACP-5b,ADAMTS-4, and MMP-3 were significantly lower in the joint intervention group than in the knee-balancing and heat-sensitive moxibustion groups after 4-week treatments and at the 2-week and 6-week follow-ups(P＜0.001). The details are shown in Table 9-Table 11.

Table 3. Comparison of the VAS score ( ±s point)

Table 3. Comparison of the VAS score ( ±s point)

Note: VAS=Visual analog scale; compared with the same group before treatment, 1) P＜0.05; compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 7.92±0.87 6.10±0.841) 6.20±0.701) 6.35±0.591)Heat-sensitive moxibustion 44 8.11±0.92 6.12±0.991) 6.22±0.691) 6.36±0.471)Joint intervention 44 8.21±0.80 5.54±0.681)2) 5.65±0.611)2) 5.88±0.651)2)F-value 0.260 50.302 10.291 9.984 P-value 0.287 ＜0.001 ＜0.001 ＜0.001

Table 4. Comparison of the WOMAC pain score ( ±s point)

Table 4. Comparison of the WOMAC pain score ( ±s point)

Note: WOMAC=Western Ontario and McMaster Universities arthritis index; compared with the same group before treatment, 1) P＜0.05;compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 5.87±1.15 2.44±1.071) 2.81±0.971) 3.14±1.331)Heat-sensitive moxibustion 44 5.69±2.06 2.49±0.851) 2.82±1.031) 3.09±1.151)Joint intervention 44 5.78±2.11 1.56±0.421)2) 2.02±1.091)2) 2.29±0.971)2)F-value 0.105 17.710 8.679 7.432 P-value 0.900 ＜0.001 ＜0.001 ＜0.001

Table 5. Comparison of the WOMAC stiffness score ( ±s point)

Table 5. Comparison of the WOMAC stiffness score ( ±s point)

Note: WOMAC=Western Ontario and McMaster Universities arthritis index; compared with the same group before treatment, 1) P＜0.05;compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 3.83±1.05 1.96±0.771) 2.21±0.881) 2.43±1.121)Heat-sensitive moxibustion 44 3.81±1.11 1.95±0.691) 2.07±0.931) 2.38±0.861)Joint intervention 44 3.85±1.3 1.03±0.571)2) 1.54±0.821)2) 1.64±0.951)2)F-value 0.013 26.960 7.085 8.898 P-value 0.987 ＜0.001 0.001 ＜0.001

Table 6. Comparison of the WOMAC daily activities score ( ±s point)

Table 6. Comparison of the WOMAC daily activities score ( ±s point)

Note: WOMAC=Western Ontario and McMaster Universities arthritis index; compared with the same group before treatment, 1) P＜0.05;compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 16.66±5.71 11.49±4.871) 12.55±3.691) 13.41±4.111)Heat-sensitive moxibustion 44 16.05±4.94 10.97±5.021) 11.97±4.331) 13.55±3.121)Joint intervention 44 15.69±5.31 8.01±2.561)2) 8.46±4.051)2) 10.63±4.331)2)F-value 0.368 8.355 13.160 7.872 P-value 0.693 ＜0.001 ＜0.001 ＜0.001

Table 7. Comparison of the WOMAC total score ( ±s point)

Table 7. Comparison of the WOMAC total score ( ±s point)

Note: WOMAC=Western Ontario and McMaster Universities arthritis index; compared with the same group before treatment, 1) P＜0.05;compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 26.77±3.57 17.33±2.831) 18.29±1.111) 19.14±3.261)Heat-sensitive moxibustion 44 25.29±3.11 18.02±2.211) 18.25±1.931) 18.95±4.141)Joint intervention 44 25.85±3.59 13.35±1.131)2) 15.27±1.281)2) 16.09±4.631)2)F-value 2.062 57.581 59.610 7.775 P-value 0.131 ＜0.001 ＜0.001 ＜0.001

Table 8. Comparison of the expression level of CTX-Ⅰ collagen ( ±s μg/L)

Table 8. Comparison of the expression level of CTX-Ⅰ collagen ( ±s μg/L)

2-week follow-up 6-week follow-up Knee-balancing 43 450.54±39.51 377.62±44.041) 388.98±34.511) 398.65±40.191)Heat-sensitive moxibustion 44 456.19±37.30 369.92±38.481) 390.02±38.771) 395.76±37.291)Joint intervention 44 455.17±29.20 297.66±38.171)2) 355.87±44.191)2) 367.89±39.381)2)F-value 0.313 52.483 10.660 8.321 P-value 0.732 ＜0.001 ＜0.001 ＜0.001

Note: CTX-I=C-telopeptide of type I collagen; compared with the same group before treatment, 1)P＜0.05; compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2)P＜0.001

Table 9. Comparison of the expression level of TRACP-5b ( ±s U/L)

Table 9. Comparison of the expression level of TRACP-5b ( ±s U/L)

Note: TRACP-5b=Tartrate-resistant acid phosphatase 5b; compared with the same group before treatment, 1) P＜0.05; compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 7.91±0.45 4.87±0.511) 5.21±0.671) 5.53±0.661)Heat-sensitive moxibustion 44 7.88±0.57 4.75±0.521) 5.35±0.291) 5.67±0.581)Joint intervention 44 7.86±0.63 3.68±0.691)2) 4.79±0.841)2) 5.10±0.831)2)F-value 0.089 55.902 9.047 7.956 P-value 0.915 ＜0.001 ＜0.001 ＜0.001

Table 10. Comparison of the expression level of ADAMTS-4 ( ±s pg/mL)

Table 10. Comparison of the expression level of ADAMTS-4 ( ±s pg/mL)

Note: ADAMTS-4=A disintegrin and metalloproteinase with thrombospondin motifs 4; compared with the same group before treatment, 1)P＜0.05; compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 267.13±11.29 240.25±11.521) 257.33±14.201) 263.17±15.351)Heat-sensitive moxibustion 44 271.30±12.54 239.15±15.671) 255.91±13.921) 267.54±10.691)Joint intervention 44 270.29±10.88 217.65±14.441)2) 245.99±15.021)2) 251.80±13.211)2)F-value 1.527 36.271 8.068 16.650 P-value 0.221 ＜0.001 ＜0.001 ＜0.001

Table 11. Comparison of the expression level of MMP-3 ( ±s pg/mL)

Table 11. Comparison of the expression level of MMP-3 ( ±s pg/mL)

Note: MMP-3=Matrix metalloproteinase 3; compared with the same group before treatment, 1) P＜0.05; compared with the knee-balancing and heat-sensitive moxibustion groups at the same time point, 2) P＜0.001

Group n Before treatment After treatment 2-week follow-up 6-week follow-up Knee-balancing 43 180.45±12.55 140.37±13.151) 152.31±10.371) 166.16±11.271)Heat-sensitive moxibustion 44 178.65±13.22 143.27±9.871) 154.25±9.991) 165.29±12.831)Joint intervention 44 181.22±13.25 127.86±12.911)2) 144.98±10.861)2) 157.58±11.741)2)F-value 0.452 20.250 9.685 6.814 P-value 0.638 ＜0.001 ＜0.001 0.002

3.3.5 Safety rating analysis

Finally, 131 cases were recruited for endpoint assessment. Their vital signs were within the normal ranges before and after treatment, including blood pressure, pulse, perspiration, heart rate, and body temperature. During the treatment, the knee-balancing group had a mild case of adverse reaction, the heat-sensitive moxibustion group had a moderate case,and the joint intervention group had one mild case and one moderate case. We found no other abnormal conditions throughout the whole study.

4 Discussion

The pathophysiological mechanism of KOA pain is not fully elucidated yet. Cartilage does not produce pain as it has no nerve endings. Therefore, in KOA, the pain usually comes from the afferent nerves of the synovial membrane, joint capsule, ligaments, and periosteum[21].Since the pain significantly hinders patients’ quality of life, it is critical to ease pain in treating KOA.

The pathological manifestations of KOA include synovial joint inflammation, formation of osteophytes,and articular cartilage degeneration. Physiological synthesis interruption and articular cartilage degeneration play crucial roles in the progression of KOA[22]. Bone turnover markers are extensively used to assess the impact of interventions on bone turnover[23].Our study found that the knee-balancing manipulation plus heat-sensitive moxibustion significantly downregulated the expression of bone absorption marker CTX-Ⅰ. The biomarker TRACP reflects osteoclast activation and bone resorption. TRACP-5b is a vital member of the TRACP family, and its serum content can evaluate bone metabolism[24]. ADAMTS-4 can damage the matrix in KOA and plays an essential role in arthritis and cartilage degeneration[25]. MMPs degrade the extracellular matrix. Increased MMPs activity is associated with cartilage degradation and plays a crucial role in articular cartilage destruction in OA patients[26].In this family, MMP-3 degrades proteoglycans and activates procollagenase, and its activation plays a key role in the development of KOA[27]. Hence, we chose these four significant biomarkers, CTX-Ⅰ, TRACP-5b,ADAMTS-4, and MMP-3, to estimate the severity of KOA in this study[28].

In this trial, we studied the efficacy of knee-balancing manipulation plus heat-sensitive moxibustion for KOA.The results demonstrated that the VAS and WOMAC scores and serum CTX-Ⅰ, TRACP-5b, ADAMTS-4, and MMP-3 dropped significantly after intervention and at the 2-week and 6-week follow-ups (P＜0.05). These scores and biomarker levels were lower in the joint intervention group than in either the knee-balancing group or the heat-sensitive moxibustion group at each time point after treatment (P＜0.001). Moreover, the total effective rate was also higher in the joint intervention group than in the other two groups at the three time points (P＜0.05). It has been proved that the knee-balancing manipulation plus heat-sensitive moxibustion can produce exact short-time and long-term efficacy with a high safety rating. This joint intervention has more significant efficacy than the two methods used separately. Next step, we should further extend the follow-up, expand the sample size, and try to elucidate the treatment mechanism with the assistance of laboratory experiments.

Conflict of Interest

There is no potential conflict of interest in this article.

Acknowledgments

This work was supported by Clinical Medical Research Project of Wuhan Health and Family Planning Commission(武漢市衛(wèi)生和計(jì)劃生育委員會(huì)臨床醫(yī)學(xué)科研項(xiàng)目,No. WZ20Q12); Major Project of National Natural Science Foundation of China (國(guó)家自然科學(xué)基金重大項(xiàng)目,No. 81590955).

Statement of Informed Consent

Informed consent was obtained from all individual participants.

Received: 19 May 2021/Accepted: 30 August 2021