Effects of fumigation and washing with Shujin Huoluo decoction combined with Duhuo Parasitic decoction on TIMP-1 and PON1 levels in KOA patients

Wei Zhou, Hong-Bo Zheng, Sui Feng, Qiang Liu, Gui-Qian Lu

Orthop Edics Department of Traditional Chinese Medicine, Liuzhou People' s Hospital, Liuzhou, Guangxi 545006, China

Keywords:Shujin Huoluo decoction fumigation and washing Duhuo Parasitic decoction addition and subtraction Knee osteoarthritis Metalloproteinase inhibitor-1

ABSTRACT Objective: To study the effect of fumigation and washing with Shujin Huoluo Decoction combined with Duhuo Parasitic Decoction on Metalloproteinase inhibitor-1 (TIMP-1) and Oxyphosphatase 1, PON1) level. Methods: A total of 100 KOA patients, 59 males and 41 females, were selected for diagnosis and treatment in our hospital from November 2018 to November 2019. The patients were divided into the control group and the combined group with 50 patients each according to a completely random method. The control group was treated with Duhuo Parasitic Decoction and the combined group was treated with Duhuo Parasitic Decoction and Shujinhuoluo decoction. Detection of TIMP-1, PON1, Matrix metalloproteinase-3 (MMP-3), C-reactive protease (CRP), Tumor necrosis factor-alpha (TNF-α) and Superoxide dismutase (SOD) levels. WOMAC score, Lysholm score, and VAS score were evaluated. Results: The levels of TIMP-1 and PON1 in the combined group were higher than those in the control group after treatment, and the levels of MMP-3 were lower than those in control group (P ＜ 0.05). The WOMAC score and VAS score of the combined group were lower than those of the control group, and the Lysholm score was higher than that of control group (P ＜ 0.05). The levels of CRP and TNF-α in the combined group were lower than those in the control group, and the SOD levels were higher than those in control group (P ＜ 0.05). The total effective rate of the combined group was higher than the total effective rate of control group (P ＜ 0.05). Conclusion: The combination of fumigation and washing with Shujin Huoluo Decoction and Duhuo Parasitic Decoction has a significant effect on the treatment of KOA patients. It improves the levels of TIMP-1 and PON1, improves the knee function of patients and pain.

1. Introduction

The incidence rate of knee osteoarthritis (KOA) is high in the middle-aged and elderly population. It is a chronic joint disease based on degenerative pathological change, hyperosteogeny of joint edge, degeneration of cartilage and narrowing of bone space. The incidence of knee osteoarthritis is obviously increased with age[1]. KOA often leads to knee joint dysfunction, pain and swelling, and the life of patients is seriously affected, even the patients with severe disability. Therefore, active and effective treatment plan is the focus of orthopaedic researchers. At present, the clinical treatment is usually based on anti-inflammatory and analgesic. In the case of alleviating the disease, long-term medication may lead to gastrointestinal side effects and kidney damage [2]. In traditional Chinese medicine, KOA disease is located in the liver and kidney, ice in the muscles and bones. If the liver and kidney are deficient, the muscles and bones are deprived of nourishment, the blood essence is lost of prosperity, and it is easy to invade the wind, cold and dampness evil [3]. Duhuo Jisheng decoction, which comes from 《Invaluable prescriptions for ready reference》, can stop arthralgia, replenish qi and blood, dispel wind dampness and benefit liver and kidney [4]. Therefore, this paper selected 100 patients with KOA who were diagnosed and treated in our hospital from November 2018 to November 2019 to study the influence of fumigation and washing of Shujinhuoluo decoction combined with the addition and subtraction of Duhuo parasitic Decoction on TIMP-1 and PON1 levels of patients with KOA.

2. Materials and methods

2.1 Materials

100 KOA patients, 59 males and 41 females, who were diagnosed and treated in our hospital from November 2018 to November 2019, were selected and divided into control group and combined group according to the complete random method, 50 cases each. The control group was 58-71 years old, the average age was (64.5 ± 8.3) years old, the course of disease was 9-28 months, the average course of disease was (18.5 ± 9.6) years old; the combined group was 57-70 years old, the average age was (64.3 ± 8.4) years old, the course of disease was 8-30 months, the average course of disease was (18.7 ± 9.5) years old. There was no statistical difference between the two groups (P ＞ 0.05).

Diagnostic standard of traditional Chinese medicine: refer to the KOA diagnostic standard in 《Guiding principles for clinical research of new drugs of traditional Chinese medicine》[5], and conform to the Yang deficiency and cold coagulation type in 《Standard of curative effect of diagnosis of diseases and syndromes in traditional Chinese Medicine》[6].

Inclusion criteria: meet the above diagnostic criteria and TCM syndrome type criteria; the patient's knee joint has bone friction and fricative sound, most of the time is painful, X-ray shows osteophyte formation, and the duration of morning stiffness is less than 30min; the patient's family members know and sign the consent.

Exclusion criteria: liver and kidney dysfunction; diseases with complications affecting the joints; rheumatoid arthritis; coagulation dysfunction.

2.2 Method

The control group was treated with Duhuojisheng decoction. The drug composition was: Duhuo 15g, Gentiana macrophylla 10g, Fangfeng 10g, asarum 5g, cinnamon 10g, Poria 10g, Eucommia 15g, Achyranthes bidentata 10g, sangjisheng 20g, prepared rehmannia 10g, angelica 10g, paeony 10g, liquorice 6g. Add and subtract: Heat syndrome removing Asarum, add Anemarrhena, gypsum; Cold syndrome add cassia twig; Add psoralen and Rehmannia to patients with tinnitus and lumbago; Add Fangji and Polyporus umbellatus to the patients with severe dampness; Add myrrh, frankincense and Corydalis to patients with long-term pain; Red and swollen joint add Gypsum and Anemarrhena. One dose per day, 400ml of water decoction, warm twice in the morning and evening.

The combination group was treated by adding and subtracting Duhuo parasitic Decoction and fumigating and washing Shujin Huoluo decoction, and the addition and subtraction of Duhuo parasitic decoction was the same as the control group: Caowu 20g, Chuanwu 20g, Eucommia 20g, mulberry branch 20g, safflower 15g, Caulis Spatholobi 20g, pepper 15g, wormwood leaf 15g, Cinnamon Twig 15g and Achyranthes bidentata 20g. Add 1.8L water to the fumigant, twice a day, fumigate for 30min each time.Both groups were treated for two weeks.

2.3 Observation index and efficacy judgment

2.3.1 Detection of TIMP-1, PON1 and MMP-3 levels

5ml of fasting venous blood was collected in the morning of the same day and placed in a disposable vacuum blood collection vessel without anticoagulant. Centrifugation was carried out at 20 ℃ - 25 ℃ for 60 min and 3000 R / min for 10 min. serum was separated and stored at - 20 ℃. The levels of TIMP-1 and MMP-3 were detected by enzyme-linked immunosorbent assay and PON1 by automatic biochemical analyzer.

2.3.2 WOMAC score, Lysholm score and VAS score detection

Assessment of osteoarthritis index using the University of Western Ontario and McMaster (WOMAC), the higher the score is, the more severe the pain is. There are 24 items in the evaluation. The painless score is 0, and the severe pain score is 10. Lysholm knee joint scoring system was used to evaluate, including 8 items, with a total of 100 points. The higher the score, the better. Using visual analogue (VAS) scoring method, the full score is 10, 0 is painless, 1-3 is mild pain, 4-6 is moderate pain, 7-10 is severe pain.

2.3.3 Detection of CRP, TNF - α and SOD levels

The levels of CRP and TNF -αwere detected by enzyme-linked immunosorbent assay, and the level of SOD was detected by chemical colorimetry.

2.3.4 Efficacy judgment

The curative effect was evaluated according to 《The standard of diagnosis and curative effect of traditional Chinese Medicine》. Significant effect: symptom score is greater than 80%, clinical symptoms and signs of patients disappear; effective: symptom score is greater than 50%, clinical symptoms and signs of patients improve; ineffective: symptom score is less than 50%, clinical symptoms and signs of patients have no obvious improvement or aggravation. Total effective rate = (number of effective cases + number of effective cases) / total cases × 100%.

2.4 Statistical treatment

SPSS 20.0 statistical software was used to analyze, t test was used to measure data, x2test was used to count data, P＜0.05 was statistically significant.

3. Results

3.1 Comparison of TIMP-1, PON1 and MMP-3 levels between the two groups before and after treatment

As shown in Table 1, there was no significant difference in the levels of TIMP-1, PON1 and MMP-3 between the two groups before treatment (P ＞ 0.05); after treatment, the levels of TIMP-1 and PON1 increased, the levels of MMP-3 decreased (P ＜ 0.05), and the levels of TIMP-1 and PON1 in the combined group were higher than those in the control group, and the levels of MMP-3 were lower than those in the control group (P ＜ 0.05).

3.2 Comparison of WOMAC score, Lysholm score and VAS score between the two groups before and after treatment

As shown in Table 2, there was no significant difference in WOMAC score, VAS score and Lysholm score between the two groups before treatment (P ＞ 0.05); after treatment, the WOMAC score and VAS score of the two groups decreased, Lysholm score increased (P ＜ 0.05), and the WOMAC score and VAS score of the combined group were lower than that of the control group, Lysholm score was higher than that of the control group (P ＜ 0.05).

3.3 Comparison of CRP, TNF - α and SOD levels between the two groups before and after treatment

As shown in Table 3, there was no significant difference in the levels of CRP, TNF -αand SOD between the two groups before treatment (P ＞ 0.05); after treatment, the levels of CRP, TNF -α and SOD in the two groups decreased (P ＜ 0.05), and the levels of CRP and TNF -αin the combined group were lower than those in the control group, while the levels of SOD in the combined group were higher than those in the control group (P ＜ 0.05).

3.4 Total effective rate of the two groups after treatment

As shown in Table 4, the total effective rate of the combined group was 94.00% higher than that of the control group (78.00%) (P ＜ 0.05).

Table 1 Comparison of TIMP-1, PON1 and MMP-3 levels between the two groups before and after treatment ( ±s )

Groups Number of cases TIMP-1（μg/L） PON1（U/L） MMP-3（ng/mL）Before treatment After treatment Before treatment After treatment Before treatment After treatment Control group 50 16.82±2.26 18.31±4.03 74.35±5.71 80.16±7.21 180.54±30.25 169.38±28.71 Joint group 50 16.79±2.24 21.57±5.34 74.26±5.68 91.72±8.13 180.61±30.27 153.17±25.06 t 0.067 3.446 0.079 7.522 0.012 3.008 P 0.947 0.001 0.937 0.001 0.991 0.003

Table 2 Comparison of WOMAC score, Lysholm score and VAS score between the two groups before and after treatment( ±s )

Groups Number of cases WOMAC score（points） Lysholm score（points） VAS score（points）Before treatment After treatment Before treatment After treatment Before treatment After treatment Control group 50 92.68±28.24 63.54±26.24 45.09±3.15 68.38±5.26 8.52±2.15 4.26±1.02 Joint group 50 92.71±28.26 46.42±24.53 45.12±3.17 75.61±6.35 8.56±2.17 2.14±0.61 t 0.005 3.371 0.047 6.201 0.093 12.611 P 0.996 0.001 0.962 0.001 0.926 0.001

Table 3 Comparison of CRP, TNF -αand SOD levels between the two groups before and after treatment ( ±s )

Table 3 Comparison of CRP, TNF -αand SOD levels between the two groups before and after treatment ( ±s )

Groups Number of cases CRP（mg/L） TNF-α(ng/L) SOD（U/mL）Before treatment After treatment Before treatment After treatment Before treatment After treatment Control group 50 10.35±3.02 6.36±1.98 271.52±40.15 165.35±35.25 82.35±3.43 98.52±5.37 Joint group 50 10.41±3.06 3.17±0.53 271.58±40.17 117.19±32.02 82.32±3.41 126.71±6.38 t 0.099 11.001 0.007 7.151 0.044 23.901 P 0.922 0.001 0.994 0.001 0.965 0.001

Table 4 Total effective rate of the two groups after treatment [n (%)]

4. Discussion

The clinical symptoms of KOA often progress slowly. Pain, stiffness, swelling and deformity of the knee joint can gradually appear over time, which can lead to the patient's inability to move flexibly and completely unable to move in severe cases [7]. KOA is the main cause of leg pain in the elderly, which is generally caused by trauma, degenerative disease and overwork. It may also be caused by cold and cold of knee joint, overweight or incorrect walking posture [8-9]. KOA belongs to the category of "osteoarthralgia", Traditional Chinese medicine believes that the joint of the disease is deficiency of vital energy, deficiency of prime body, invasion of cold and dampness into knee joint, and caused by unstable external health [10]. The main manifestations of KOA are dampness heat obstruction, wind cold dampness obstruction, deficiency of stomach, liver, spleen and kidney, qi stagnation and blood stasis, Traditional Chinese medicine treatment can relax the muscles and activate the bones, dispel the cold and relieve pain, strengthen the muscles and bones, benefit the spleen and kidney, and effectively promote the recovery of metabolism balance of the knee joints [11].

In the prescription of Duhuo parasitic decoction, Duhuo can dispel the pathogenic wind cold and dampness between the muscles and bones, and can relieve the arthralgia and pain; Gentiana macrophylla can remove rheumatism, stop arthralgia, relax muscles, collaterals and joints, and has the effect of anti inflammation and detumescence; Fangfeng can dispel wind to overcome dampness, dredge meridians, meridians and joints, relieve spasm and pain, and improve limb function; Asarum has the effect of dispersing Yin meridians and wind chill, strengthening muscle and relieving pain; Cinnamomum cassia has a hot nature, which can play the role of dispersing cold and relieving pain, promoting blood circulation, warming yang and invigorating the kidney; Poria cocos has the functions of invigorating the spleen and removing dampness, Invigorating qi and reducing swelling; Eucommia, Achyranthes bidentata and mulberry parasite can nourish the liver and kidney, strengthen the muscles and bones. Achyranthes bidentata can promote blood circulation and remove blood stasis, clear the joints, and mulberry parasite can also disperse cold and dehumidify; Radix rehmanniae can promote the proliferation and differentiation of hematopoietic stem cells, and play the role of Tonifying the kidney, nourishing the essence and filling the marrow; Angelica can promote blood circulation and nourish blood, and paeony can protect liver and relieve pain; Liquorice mixed with various medicines, this prescription can remove pathogenic factors and strengthen the body, and give consideration to both the specimen and the blood circulation, nourishing yin and kidney, relaxing muscles and activating collaterals, and eliminating dampness and pain [12-13].

The expression of TIMP-1 in normal joints is very low. Abnormal expression of TIMP-1 in patients with koa can destroy the degradation of type I collagen in the peripheral tendon and intraarticular ligament, can cause instability of the joint, thus increasing the degradation of cartilage [14-15]. PON1 is an important component of anti-oxidation mechanism, which can effectively prevent HDL and LDL from being damaged by oxidation, prevent LDL from being oxidized, and reduce the oxidation pressure in the joint cells. Therefore, PON1 is one of the important factors to evaluate the condition of knee osteoarthritis [16]. The results showed that the levels of TIMP-1 and PON1 in the combined group were higher than those in the control group, and the levels of MMP-3 were lower than those in the control group. The results showed that Shujinhuoluo decoction combined with duhuojishi decoction can significantly improve the TIMP-1 and PON1 levels and reduce the MMP-3 level in patients with KOA. In this study, the WOMAC score and VAS score of the combined group were lower than those of the control group, and the Lysholm score was higher than that of the control group. It is suggested that Shujinhuoluo decoction combined with Duhuojisheng decoction can improve the pain and knee function of KOA patients. The activity of SOD can evaluate the scavenging ability of oxygen free radicals to a certain extent, which is an important antioxidant enzyme [17]. This study shows that the levels of CRP and TNF -αin the combined group are lower than those in the control group, and the level of SOD is higher than that in the control group. It is suggested that Shujinhuoluo decoction combined with Duhuojisheng decoction can reduce the inflammation of KOA patients.

Radix Aconiti Kusnezoffii Preparata and Radix Aconiti Preparata in Shujin Huoluo decoction can warm meridians, relieve pain, expell wind and dehumidification; Eucommia strengthens muscles and bones, nourishes liver and kidney; Mulberry branch can dredge channels and collaterals, benefit joints and relieve arthralgia; Honghua dispersing blood stasis and relieving pain, promoting blood circulation and promoting meridian circulation; Caulis Spatholobi can promote blood circulation and invigorate blood, relax muscles and activate collaterals; Chinese prickly ash chases cold, acesodyne; artemisia argyi can disperse cold to relieve pain, warm meridians to stop bleeding; Guizhi helps promote Yang and transform Qi, warm meridians and channels; Achyranthes bidentata is strong in muscle, bone and blood stasis. The results showed that the total effective rate of the combined group was higher than that of the control group. It shows that Shujinhuoluo decoction combined with Duhuojisheng decoction has a significant therapeutic effect on patients with KOA. Duhuoparasitic decoction can effectively improve the non-specific immune function of patients, play the role of analgesia and antiinflammatory, expand blood vessels, regulate immune balance, and improve circulation [18-19]. Shujin Huoluo decoction can remove blood stasis, invigorate liver and kidney, relieve pain, disperse wind and cold, smooth joints, activate blood and tendons [20].

In conclusion, Shujinhuoluo decoction combined with Duhuojisheng decoction can significantly improve the level of TIMP-1 and PON1, improve the knee joint function, reduce the pain and inflammatory response of patients, which is worthy of clinical promotion.