Effect of Baihu and Guizhi Decoction in acute gouty arthritis: a randomized, double-blind, controlled clinical study

The prevalence rates of gout worldwide have increased annually. Acute gouty arthritis (AGA) accounts for a large proportion of gout patients and causes severe physical and mental pain in patients. Controlling the occurrence and development of gout inammation is the rst step in the treatment of gout. The main treatment drugs in gout are Non-Steroid Anti-Inammatory Drugs (NSAIDS), colchicine and glucocorticoids, but these treatments have many adverse reactions which limit their clinical application. Baihu and Guizhi decoction (BHGZ) is one of the classic prescriptions in the Synopsis of the Golden Chamber, and is a good prescription for AGA. Previous clinical studies have shown that BHGZ confers a strong benet for treating AGA. However, the literature shows a lack of high-quality RCT research on BHGZ with respect to AGA. Therefore, in this study, we use a randomized, double-blind, controlled study with a placebo to evaluate the clinical ecacy and safety of BHGZ on the AGA of moist heat arthralgia spasm syndrome.

Gout is a metabolic in ammatory disease caused by the deposition of monosodium urate (MSU) in the cartilage, synovial sac, tendon, soft tissue and/or kidney. Gout is directly related to hyperuricemia, potentially attributable to changes in lifestyle and diet. The prevalence rates of gout worldwide have increased annually [1]. In the USA, the morbidity of gout is 3.76%, and in Britain, it is 2.49% [2]. The prevalence of gout in China is 1-3% and shows an increasing trend, particularly among younger people, with lower incidence in inland than in coastal areas and a higher prevalence in males than in females [3].
The development of gout includes four stages: asymptomatic hyperuricemia, acute attack, remission and chronic gouty arthritis (gout stones). An acute attack of gout is closely related to the rapid uctuation of serum uric acid. In acute attacks, the rapid development of severe pain, swelling, and tenderness can reach its maximum within 6-12 h of the immune response being triggered by urate. Thus, the early treatment of acute gouty arthritis (AGA) not only quickly improves the clinical symptoms but also reduces the in ammation and joint tissue damage mediated by the immune response. The aim of AGA treatment is attenuated in ammation and analgesic relief, and the main treatment drugs are Non-Steroid Anti-In ammatory Drugs (NSAIDS), colchicine and glucocorticoids. The American College of Rheumatology (ACR) criteria and European League Against Rheumatism (EULAR) guidelines of 2015 also recommended the use of these three types of drugs [4]. However, these drugs are associated with many adverse reactions, such as colchicine-induced diarrhea and liver and kidney toxicity; NSAID-induced gastric mucosa damage and liver and kidney injury; and glucocorticoid-induced metabolic disorders and osteoporosis. These adverse reactions greatly limit their clinical application, particularly for patients with liver and renal insu ciency and acute gout. Therefore, it is necessary to explore additional rapidly effective and less adverse drugs in the treatment of AGA.
Chinese herbal medicine has been used in China for thousands of years. Syndrome differentiation and treatment are the main characteristics and therapeutic guidelines for traditional Chinese medicine (TCM). According to the diagnostic criteria of TCM, the analysis of clinical data is obtained from the four main diagnostic methods-observation, listening, interrogation, and pulse-taking. After years of clinical practice treating AGA, most traditional Chinese medicine scholars believe that moist heat arthralgia spasm syndrome is the common TCM syndrome of AGA [5]. The clinical manifestations are as follows: swollen small joints of the lower limbs, painful and heated acute attacks relieved by coolness and prevention of added pressure, fever, thirst, sweating, yellow urine, red tongue with yellowish or greasy fur, and rapid pulse. The Baihu jia Guizhi decoction (BHGZ) from the Synopsis of the Golden Chamber can clear away heat, remove dampness, dredge collaterals and relieve pain. It is a classic prescription for the treatment for the moist heat arthralgia spasm syndrome of gout in Traditional Chinese Medicine Internal medicine published by China Press of Traditional Chinese Medicine [6]. Previous clinical studies have shown that BHGZ confers a strong bene t for treating AGA [7][8]. However, the literature shows a lack of high-quality RCT research on BHGZ with respect to AGA. Therefore, in this study, we use a randomized, double-blind, controlled study with a placebo to evaluate the clinical e cacy and safety of BHGZ on the AGA patients of moist heat arthralgia spasm syndrome. We hypothesized that BHGZ can effectively reduce the clinical symptoms and signs, improve quality of life, shorten the treatment time, quickly relieve joint in ammation, and demonstrate a good safety pro le for use on AGA patients of moist heat arthralgia spasm syndrome.

Methods
This is a randomized, double-blind, controlled exploratory study with a 10-day follow-up. Each participant will be asked for written informed consent ( Figure 1).

Sample size calculation
To determine the sample size, we used the ndings from previous studies. The rate of colchicine effectiveness in the treatment of AGA was 67% [9]and that of BHGZ was 91% [8]. When α = 0.05 and the test e cacy 1-β = 0.80, the sample size needs to be n=81, with 41 in each treatment group. Considering a 20% dropout rate, the planned sample size for randomization was increased to 102. The formula for calculation is as follows: n= U α +U β 2 1+1/k P 1-P / P 1 -P 2 2 This study will recruit participants from Shuguang Hospital a liated with Shanghai University of TCM. An advertisement encouraging clinical trial enrollment will be posted on the webpage. We plan to recruit patients from participating hospitals via poster, networking, or WeChat. The posters will be placed on bulletin boards or other assigned places in the hospitals. At least one staff member (a postgraduate or doctor) will specialize in patient recruitment. Their contact information and clinic for screening visit will be detailed described in the recruitment advertisement.
Diagnostic criteria for AGA The diagnosis of acute gout arthritis is made according to the 2015 ACR criteria and EULAR Classi cation Criteria for AGA [4].

Diagnostic criteria for TCM syndrome differentiation
The diagnosis of " moist heat arthralgia spasm syndrome" is based on the following diagnosis and Swollen small joints of the lower limbs and painful acute attack and heat which are relieved by application of coolness and prevention of added pressure, accompanied by fever, thirst, upset and yellow urine .
Tongue and pulse: red and rolling tongue with yellow or greasy fur and rapid pulse.
Inclusion criteria 1. Adult subjects with a diagnosis of AGA as stated above and with a TCM diagnosis of moist heat arthralgia spasm syndrome. 3. Allergies to the tested drugs.
4. Abnormal liver function: an ALT level that is higher than twice the normal level.
5. Severe acute/chronic organic or mental diseases.
. Severe deformity or inability to be in the labor force because of late-stage arthritis.
7. Poor compliance and/or inability to complete the clinical observation.

Rejection criteria
1. Failure to meet the criteria but mistakenly admitted.
2. Failure to take medicine according to instruction during the trial, affecting the e cacy results.
3. Patients who take other traditional Chinese medicine drugs that are prohibited by the instructions of the trial, interfering with the correct evaluation of the e cacy and safety.

Randomized and blind review
The randomization was computer generated by an independent statistician using SPSS software (SPSS version 21.0; IBM, NY, USA). Participants will be randomly assigned (on a 1:1 ratio) to a BHGZ group and placebo group. If the patients agree to participate and sign the informed consent voluntarily after screening, then an independent research physician, who is not engaged in the recruitment for this study, the treatment provided, or the assessments will assign a random sequential number to the participant.
The packaging of the BHGZ or placebo drug will be labeled with the random sequential number according to a random code. The independent statistician will examine and verify the packaging of the drugs and placebo by the pharmaceutical company during labeling. Each patient will be distributed a 3-day dose with the lowest number drugs or placebo by a clinical pharmacist, who does not know the allocation groups of the participants.

Blind review
The trial will use the double-blind, placebo-controlled method. The BHGZ granules and placebo granules have the same size, shape, color and packaging. An independent statistician will divide the BHGZ and placebo drugs into two types (A and B) and then seal the information to the bottom of the packaging, according to the random results. This blind method will be applied to both participants and researchers (including the physicians, investigators, coinvestigators and pharmacists), and the drugs were distributed according to the visiting sequence. Unblinding will be performed according to the standard operating procedure (SOP) of the Contract Research Organization (CRO) [10]. If adverse events occur during the test, then the principal investigator will be given an emergency envelope for to use as a backup, and a secondary blind method will be used, with the principal investigator reporting this process to the independent safety monitoring board. When the trial is completed, the independent statistician will lock the data after checking to ensure that the data cannot be modi ed. Next, the independent statistician who preserved and sealed the information to the bottom of the packaging will start the rst level of unveiling and immediately inform the statistician about the corresponding group numbers for each enrolled patient. The statistician will conduct statistical analysis on all the collected data. After the completion of the statistical analysis of the data, the blind information will be revealed again. Finally, the groups corresponding to A or B will be announced by the independent statistician.
Intervention Basic therapy Two groups will follow a basic Western medicine treatment plan as indicated by the suggested use of colchicine in the Chinese gout diagnosis and treatment guide issued by the rheumatology branch of the Chinese Medical Association in 2016 [11]: A colchicine tablet taken orally (Kunming Pharmaceutical Group Co., Ltd.; 1.0mg/tablet; H53021534) as follows: 1.0 mg initially, then 0.5 mg one hour later, then 0.5 to 1.0 mg per day. If the participant has taken an uric-acid-lowering drugs drug before entering the group, the drug and drug dosage will remain unchanged.
Test drug name and speci cation The BHGZ granules comprise gypsum (Shigao; 30 g), Rhizoma Anemarrhenae (Zhimu; 9 g), prepared Radix Glycyrrhizae (Zhi Gancao; 3 g), raw Chinese yam (Sheng Shanyao; 6 g) instead of rice (Jingmi), and cassia twig (Guizhi; 5 g). The placebo comprises starch, dextrin, and bitter agent, which contain onetenth of the content of the BHGZ. Its smell and taste are similar to those of the BHGZ granules. The BHGZ granules and the placebo granules are both manufactured by Jiangyin TianJiang Pharmaceutical Co., Ltd., and are used for the preparation of traditional Chinese medicine formula granules.
The composition and e cacy of each herb in the BHGZ granules are summarized in Table 1.

Outcome measures and time points
The general information, symptoms and signs of the subjects will be collected at baseline (the day of enrollment) and on the 3rd, 7th and 10th day in the treatment, and the relevant examinations will be carried out at the baseline and on the 10th day of treatment.
The general information includes the subjects' age, gender, nationality, occupation, ID number, contact information, medical history, allergy history, family history and breath, body temperature, pulse, blood pressure, tongue coating, and pulse. The symptoms and signs will be measured using a VAS test, classi cation of joint tenderness, joint swelling and extent of joint movement disorder, and TCM syndrome curative effect score. The relevant examinations include laboratory tests and musculoskeletal ultrasonographic examinations. The laboratory tests include measurements of xanthine oxidase (chemical colorimetry) and serum uric acid (SUA, with an automatic biochemical analyzer), albumin/creatinine ratio, erythrocyte sedimentation rate (ESR, by the automatic ESR method), C-reactive protein (CRP), and levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1, IL-10 (by ELISAs), NLRP3, nuclear factor kappa-B (NF-κB) and caspase-1 (by RT-PCR), These indicators will be measured at the Shuguang Hospital Medical Testing Center.
Musculoskeletal ultrasound will be performed by two experienced doctors. To reduce bias, the ultrasound doctors will be blinded to the clinical examination and laboratory results and prohibited from inquiring about a patient's clinical symptoms during the examination. High-frequency ultrasound will be used to judge the synovial lesions in both knees, ankles and rst metatarsophalangeal joint of both feet. The synovial thickness, blood ow signal, resistance of blood ow signal and double track sign will be measured. Each image will be saved, and the grade and total score of the detected lesions will be recorded.
The measured items and time points of data collection are found in Table 2.

OUTCOMES
The primary outcomes of this trial will include the improvement of joint symptoms and signs (VAS test, joint tenderness, joint swelling and joint movement disorder), ESR, CRP and evaluation of the joints via musculoskeletal ultrasound. Secondary outcomes will include changes in TCM symptoms; xanthine oxidase, 24-h urine uric acid, and 24-h urine creatinine levels; the uric acid excretion rate, FEua; ACR; and SUA, TNF-α, IL-6, IL-1, IL-10, NLRP3, NF-κB and caspase-1 levels. Composite outcomes will include the incidence rates of all the possible safety events in the two groups. AEs will be monitored throughout the trial, and several biological indicators (blood, urine and stool tests; routine liver and kidney function tests; and ECG tracings) will also be closely monitored.

Measurement scale for TCM symptoms
To facilitate the evaluation of the results, we will use the measurement scale for TCM symptoms recommended by the Guidelines for Clinical Research of Chinese Medicine (New Drug) [12]to score the symptoms of the patients. The scale is based on 4 major symptoms and 7 minor symptoms. Each symptom is graded on 4 levels: as none, light, medium and heavy with corresponding scores of 0, 1, 2 and 3, respectively. The total score was calculated by adding each score in the scale to calculate an e cacy indicator (EI) that is used to evaluate treatment e cacy: EI = (Total symptom score at baseline -Total symptom score post treatment)/Total symptom score before treatment×100%.
Evaluation of joint symptoms and signs 1. The visual analog scale (VAS) is presented on a 10-cm line with anchor statements on the left (no pain) and on the right (extreme pain).
The patient is asked to mark their current pain level on the line. They may also be asked to mark their maximum, minimum, and average pain. The examiner scores the VAS by measuring the distance in either centimeters (0 to 10) or millimeters (0 to 100) from the "no pain" anchor point.
0 points: A distance of less than 1 cm indicates the current pain level.
1 point: A distance between 1 and 4 cm indicates mild pain but with the ability to engage in daily activities.
2 points: A distance between 4 and 7 cm indicates moderate pain with daily activities being affected.
3 points: A distance between 7 and 10 cm indicates severe pain in daily life with restricted daily activities.
2. Evaluation of joint tenderness 0 points: No pain when under heavy pressure or performing passive activities.
1 point: Mild pain: the patient claims tenderness with heavy pressure on the edge of the joint or with touch, but passive movement is not restricted.
2 points: Moderate pain: The patient can feel pain with heavy pressure and expresses discomfort. Passive movement is sometimes restricted.
3 points: Severe pain: The patient can feel pain with heavy pressure and tenderness and retreat. Passive mobility is severely restricted. 3 points: The joints are severely swollen, and the skin is tight with the loss of bone marks.

Evaluation of joint mobility
1 point: Activities are slightly restricted, but the patient can participate in normal activities.
2 points: Obvious restricted activities. Normal activities are not permitted, but patient can perform self-care.
3 points: Activities are severely restricted, and the patient and cannot perform self-care.
(5) Evaluation criteria of the synovial thickness and blood ow signal by musculoskeletal ultrasound.
The synovial thickness and blood ow signal are graded using the semi-quantitative method a. The synovial thickness is divided into 4 grades [13]: Grade 1: no synovial hyperplasia with < 2-mm thickness.
b. The blood ow signal levels are divided into 3 grades [14].
Grade 0: no signal, no blood ow.
Grade 2: medium, fused vessels; > 3 independent signals or less than one-half of the synovial region.
Grade 3: signi cant vascular signals are observed in more than one-half of the intra-articular area.

Protection of the subjects' rights
The informed consent form was formulated by the requirements of the "Declaration of Helsinki" and "Management Practices for Drug Clinical Research". We will provide subjects with detailed information about the clinical study, including the research objectives, research methods and processes, such as treatment measures, grouping, testing, the expected bene ts for the subjects, possible risks and inconveniences. At the same time, the subject's personal information remains con dential. The subject's participation in the clinical study is completely voluntary. At any stage of the clinical study, a participant can withdraw from the clinical research without discrimination or retaliation. Patient rights and interests are not affected. If harm or injury related to the clinical research occurs, then the subjects can be appropriately compensated. The clinical study can be conducted only after the signature of the subject or legal representative/guardian is received with the date speci ed. If the subject or legal representative/guardian cannot read, a witness should be present. After a detailed explanation of the informed consent form, the subject or legal representative/guardian can provide consent, and the witness can sign and date the form.

Adverse events/Serious adverse events
We will monitor the adverse events of each treatment during the trial, including acute pain and gastrointestinal discomfort. Any adverse events or reactions that are believed to be causally related to the intervention will be recorded, managed, and reported to the study coordinator. Serious adverse reactions will be reported to the ethics committee.
Data quality control A training will be done for all participating staff on the trial protocol, usage of the randomization, and data management systems, etc. The principle investigator will supervise the proceed of the trial at least once every month. An ethics committee will review conduct especially on safety, rights, and well-being of the participants at the middle and the end of the trial. The auditing will be done by Clinical Evaluation Center of the China Academy of Chinese Medical Sciences at the beginning, middle, and end of the trial.

Data collection and management
Composition of data monitoring committee (DMC) is composed of professional statisticians. They will perform statistical analysis, participating in the entire process from the research design and implementation to the analysis and summary. A statistical analysis plan will be developed after the completion of the study program and completion of the case reporting forms, and the statistical analysis reports will be provided after the necessary modi cation of the data analysis is performed as necessary during the research process. According to the project of the case report form, EpiData3.1a software will be used to establish the corresponding entry procedure and set the logical examination quali cations at the time of entry, and the database will be piloted to establish a database system dedicated to this experiment. The signed case report form and the audit statement will be given to the data administrator, who will examine the date, group criteria, culling criteria, shedding criteria, and missing values. If there is doubt about "data question form", it will be returned to the monitor, and the researcher will answer and sign the question in writing and return it to the data administrator; "data question form" should be properly stored to protect con dentiality before, during, and after the trial. The data are entered synchronously by the data administrator using a two-person entry method. The database will be checked for each item using the veri cation function in the EpiData3.1a software; any inconsistent result values will be reported. The original case reporting tables will be checked item by item, and 10 case reporting tables and the data in the database will be randomly selected for manual comparison to ensure that the data in the database are consistent with the results in the case reporting tables. The original CRFs and any other records will be archived for 5 years.

Statistical analysis
According to the intention to treat (ITT) principle, all the selected patients will be included in the preliminary analysis. The study will use SPSS 21.0 statistical software (SPSS Inc., Chicago, IL, USA) for statistical data analysis. If the measurement data follow a normal distribution, t-tests will be used for analysis, and the results will be expressed as the means ± standard deviation ( ± s). If the data do not follow a normal distribution, Mann-Whitney U tests will be used, and the results will represent the medians and quartile M (P25-P75) values. For the measurement of data at multiple time points (>2) that follow a normal distribution, repeated-measurement data analysis of variance will be used. The measurement data at multiple time points (>2) that do not follow a normal distribution will be subjected to a generalized estimation equation. Chi-square (c 2 ) tests will be used for the numeration data. Mann-Whitney U tests will be used for the grade data. For the analysis of the level data at multiple time points (>2), a generalized estimation equation will be adopted, and P<0.05 will be considered statistically signi cant. We will also perform strati ed analysis to control confounding factors if necessary. Data analysis will be performed by statisticians independent of the research team.

Ethics and dissemination
This trial protocol will be performed according to the principles laid down in the Declaration of Helsinki, has been approved by the ethics committee of Shuguang Hospital A liated with Shanghai University of Traditional Chinese medicine (approval no. 2018-617-46-01) and is registered at the Chinese Clinical Trial Registry (ChiCTR1900024974).
A quali ed doctor will conduct the study as the principal investigator. The subject's personal information and all the data in the trial will not be disclosed to any third party. The patients will be included in the study after receiving the study information and signing the informed consent form. The research results will be published in a peer-reviewed journal and will be disseminated in open-access, peer-reviewed journals and will be shared through oral and poster presentations at domestic conferences. All the resources will be uploaded to an online knowledge management platform. Plans for communicating important protocol modi cations to researchers in time.

Discussion
AGA is a crucial stage in the development of gout. As the deposition of monosodium urate (MSU) exceeds the normal threshold, the in ammatory reaction of the joints is rapidly increased, AGA will occur with the features of acute onset. AGA accounts for a large proportion of gout patients and causes severe physical and mental pain in patients [15]. Controlling the occurrence and development of gout in ammation is the rst step in the treatment of gout.
Colchicine has been o cially used as the treatment of AGA since the 13th century and remains one of the most-recommended rst-line drugs to treat AGA in many clinical guidelines. It is a classic and speci c drug for AGA that can attenuate in ammation and relieve pain. Many studies [16-18] have con rmed that low-dose and high-dose colchicine can effectively reduce the pain of a gout attack, and the side effects of low-dose colchicine are better than those of high-dose colchicine. Therefore, in recent years, several worldwide gout guidelines [19][20] have recommended low-dose colchicine or its combination with NSAIDs as treatment for AGA. However, some patients taking small doses of colchicine have gastrointestinal reactions, such as diarrhea and nausea, and it may not control severe joint pain. Some patients limit the use of this drug because of liver and kidney damage [21][22]. Thus, increasing the effectiveness or reducing the toxicity of colchicine, or even identifying an alternative, is di cult in the treatment of AGA.
Most traditional Chinese medicine scholars believe that gout patients mostly consume excess alcohol and greasy and surfeit avored foods, causing dampness that blocks the middle-jiao, transform heat and accumulation, dampness and heat accumulation in the joints, blocked blood vessels and joint swelling and pain. Thus, moist heat arthralgia spasm syndrome is a common TCM syndrome type of AGA. AGA belongs to the category of "arthralgia syndrome" of TCM, for which the moist heat arthralgia spasm syndrome BHGZ is a commonly used prescription. Many studies have used BHGZ in the treatment of AGA, and the curative effect is signi cant [23][24][25]. However, a search of the literature from the past 20 years revealed that most of the current treatments of AGA are based on BHGZ with components added or omitted. Although these studies showed the characteristics of TCM syndrome differentiation, they did not re ect the curative effect of the BHGZ, and the reference and comparability between the studies were poor. Additionally, there was a lack of high-quality RCT studies to evaluate the clinical e cacy of the BHGZ on AGA. Therefore, in this study, BHGZ decoction granules will be used in the clinical treatment of AGA with a strict randomized, double-blind, placebo treatment method used to observe the clinical e cacy of the prescription for patients with the damp heat obstruction type of AGA. quickly relieve joint in ammation. We expect that this trial will provide preliminary evidence for the e cacy of BHGZ in treating AGA patients. It will also provide substantial help for researchers, practitioners and patients.
This study possesses some limitations. First, no international uniform standard is currently available for the musculoskeletal ultrasound evaluation of AGA in patients. Second, the study will be conducted in Shanghai, China. It remains uncertain whether the relative effects of BHGZ will be similar in other ethnic groups. Third, the speci c components in BHGZ that affect the therapeutic response will require further research and exploration.

Trial Status
The protocol version number is V2.0,and the research strategy and study protocol were developed between October 2018 and June 2020. The follow-up visits and data analysis will take place from July 2020 to December 2020.

Tables
Please see the supplementary les section to view the tables. Figure 1 Study ow chart.