Application of ARFI technology to explore the clinical study of Gandou Tang in treating liver fibrosis of wilson's disease with damp-heat accumulation

Han Hui, Wang Mei-xia, Tao Zhuang, Zhang Yanyun, Zhang Yu, Jiang Zhenzhen,Wang Fei, Sun Kuo

1.The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China

2.The Graduate School of Anhui University of Traditional Chinese Medicine, Hefei 230038, China

Keywords:

ABSTRACT Objective: Acoustic radiation force impulse (ARFI) was applied to measure Shear wave velocity (SWV) of liver in patients with Wilson's disease (WD).To investigate the relationship between SWV and the serological indexes of liver fibrosis, such as type Ⅳ collagen(CⅣ), hyaluronic acid(HA), type Ⅲ procollagen peptide(PⅢNP), laminin(LN)), APRI score(Asparate Aminotransfer to Platelet Ratio Index), and FIB-4 index (FIB-4 index).The clinical efficacy of GandouTang (GDT) in the treatment of liver fibrosis in WD with damp-heat internalization was also observed.Methods: 80 cases of WD patients who met the inclusion criteria were randomly divided into the treatment group and the control group, with 40 cases in each group.The control group was treated with Sodium Dimercaptopropylsulfonate(DMPS)and the treatment group was additionally treated with the traditional Chinese medicine GDT.One course for 8 days, a total of 6 courses.The levels of SWV and four serological indicators of liver fibrosis (PⅢNP, HA, CⅣ, LN), APRI score and FIB-4 index were compared before and after treatment.Pearson correlation test was used to analyze the correlation between SWV and HA, CⅣ, LN, PⅢNP, APRI score and FIB-4 index.The effects of GDT on SWV,liver fiber, APRI and FIB-4 were evaluated according to the treatment plan.Results: ①The SWV was positively correlated with FIB-4(r=0.83), APRI(r=0.82), HA(r=0.87), CⅣ(r=0.71),LN(r=0.85) and PⅢNP(r=0.77).②Before treatment, there were no significant differences in SWV level, PⅢNP, HA, CⅣ, LN, APRI and FIB-4 levels between two groups (P＞0.05).After treatment, the levels of PⅢNP, HA, LN, SWV, APRI and FIB-4 in both groups were significantly decreased (P＜0.05, P＜0.01), and the levels in the treatment group were lower than those in the control group.There were no significant specific changes in CⅣ level(P＞0.05).Conclusion: SWV value can reflect the degree of WD liver fibrosis, and is positively correlated with HA, PⅢNP, CⅣ, LN, FIB-4 index and APRI score.On the basis of the treatment of protecting liver and expelling copper with western medicine, plus the treatment of traditional Chinese medicine GDT can effectively improve the degree of liver fibrosis in WD patients with damp-heat accumulation.

1.Introduction

Hepatolenticular degeneration is also called Wilson’s disease(Wilson’s disease, WD), which is a recessive genetic disease caused by ATP7B gene mutation on the autosomal, resulting in abnormal copper metabolism of the body, thus damaging the organs of the whole body,Clinical common liver, brain, kidney and other human organ damage[1-3].Liver fibrosis is an important manifestation of liver injury in WD patients.Clinical research shows that[4] liver fibrosis can be reversed by effective treatment.The diagnosis of liver fibrosis by liver puncture is invasive and difficult to repeat,so the non-invasive diagnosis technology of liver fibrosis has attracted more attention in recent years.Acoustic radiation force pulse (ARFI) is a simple and repeatable non-invasive method for evaluating liver fibrosis, which has good diagnostic value for liver fibrosis and cirrhosis.Therefore, the application of ARFI technology has important clinical significance[5-6].

The study showed that[7-8], the shear wave velocity (SWV) of the liver obtained by ARFI combined with APRI score (Asparate Aminotransfer to Platelet Ratio Index), FIB-4 index (FIB-4 index)and four types of collagen type Ⅳ (CIV), hyaluronic acid (HA), typeⅢ procollagen peptide (PⅢNP) , laminin (LN) can significantly improve the diagnostic rate of liver fibrosis.

The Department of Encephalopathy of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, as the diagnosis and treatment center of hepatolenticular degeneration, a national key disease of traditional Chinese medicine, has a significant clinical effect in the treatment of WD with traditional Chinese medicine.Previous studies have shown that[9-10] GDT has the effects of clearing heat and detoxicating, and removing dampness.Therefore,on the basis of previous studies, this study selected patients with hepatic fibrosis of WD with damp-heat accumulation, dynamically monitored the level of SWV, and discussed the correlation between SWV and the serological indicators of liver fibrosis, HA, C Ⅳ, LN,PⅢ NP, APRI score and FIB-4 index, so as to reflect the change of the degree of liver fibrosis, and provide a reference for the clinical efficacy of GDT in treating WD hepatic fibrosis.

2.Data and methods

2.1 General information

From August 2021 to August 2022, 80 patients with damp-heat intrinsic WD in the Department of Encephalopathy of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine were collected and divided into treatment group and control group by random number table, with 40 patients in each group.In the treatment group, there were 22 males and 18 females,aged (15-57) years, with an average age of 32.2 ± 10.24 years and an average course of disease of 8.32 ± 6.72 years.In the control group,there were 20 males and 20 females, aged (16-55) years, with an average age of 31.63 ± 9.18 years; The average course of disease was 8.50 ± 5.32 years.There was no significant difference between the general data of each group (P＞0.05).

2.2.Diagnostic criteria

2.2.1 WD diagnostic criteria

It is formulated with reference to the Guidelines for the Diagnosis and Treatment of Hepatolenticular Degeneration[11] formulated by the Chinese Medical Association in 2008.

2.2.2 Diagnostic criteria of WD damp-heat syndrome

Referring to Professor Yang Wenming’s Diagnosis and Treatment Plan for Hepatolenticular Degeneration[12], the diagnostic criteria of WD damp-heat syndrome are: tremor of hands and feet, vague speech, difficulty in walking, stiff contracture of limbs, bitter mouth and bad breath, persistent salivation, epistaxis, jaundice, bloating,fullness of abdomen, red urine, constipation, red tongue, yellow and greasy fur, and pulse string slip number.

2.2.3 Diagnostic criteria for WD liver fibrosis

At present, clinical diagnosis of liver fibrosis can be made by liver biopsy.When the degree of fibrosis is more than F2, liver fibrosis can be diagnosed.Because liver biopsy is a invasive examination,patients can also be detected by ARFI technology.ARFI reaches the hardness value of liver fibrosis and can be diagnosed as liver fibrosis[13].

2.3.Inclusion criteria

①Conform to the diagnostic criteria of WD western medicine and the characteristics of TCM syndrome classification;②It also meets the diagnostic criteria for liver fibrosis;③All patients were initially diagnosed or did not receive standard copper removal treatment within half a year;④The subjects understood the purpose of the study, volunteered to participate in the study and signed the informed consent form (minor patients need the guardian’s consent and signed the informed consent form).

2.4 Exclusion criteria

①Pregnancy and lactation patients;②Patients with autoimmune liver disease;③Those with infectious diseases and immune diseases;④Those with severe mental disorder and other serious cardiac and renal function impairment;⑤Asymptomatic WD patients;⑥Incomplete clinical data;⑦Poor compliance.

2.5 Treatment

2.5.1 Control group

Sodium dimercaptopropanesulfonate (DMPS, Shanghai Hefeng Pharmaceutical Co., Ltd., GYZZ H31021514) was administered intravenously.Dissolve 20mg/kg of DMPS in 250 mL of 5%glucose solution for intravenous drip for 6 consecutive days, with an intermittent period of 2 days.During the intermittent period, 20 mL of 10% calcium gluconate is intravenous drip every day.This 8 days is a course of treatment, a total of 6 courses of treatment.

2.5.2 Treatment group

On the basis of the control group, GDT (prescription: scutellaria barbata, scutellaria baicalensis, andrographis paniculata, raw rhubarb,coptis chinensis and bixie) was given.All the pieces were from the Pharmacy Department of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, and the decoction was prepared by the Preparation Center of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, one dose per day(about 200 mL), taken separately in the morning and evening, 8 days as a course of treatment, a total of 6 courses of treatment.

2.6 Observation index and curative effect judgment

(1) Liver shear wave velocity (SWV): The color Doppler ultrasound diagnostic instrument (SIEMENS Acuson S2000) with 4C1 convex array probe is equipped with ARFI imaging system,and its frequency range is 1.5～4.0 MHz.First, instruct the patient to lie flat, perform routine ultrasound scanning and color blood flow imaging of the liver on an empty stomach, then instruct the patient to lie on the left side, raise the right upper limb, fully expose the liver area, place the probe at the front of the right armpit to the 7th to 9th intercostal space of the midaxillary line for scanning, pay attention to avoiding the large blood vessels and bile duct structures in the liver (such as portal vein, hepatic arteriovenous and biliary system),and instruct the patient to hold his breath and start the ARFI imaging system, Measure the shear wave velocity (SWV) of the liver, repeat for 5 times, and take the average of the 5 measured values.

(2) Laboratory examination: peripheral venous blood was taken before and after treatment.The serum indexes of liver fibrosis were determined by radioimmunoassay: HA, LN, PC Ⅲ, C-Ⅳ(batch number: 20190409,20190412,20190427,20190407,Zhen gzhou Antu Bioengineering Co.,Ltd.).Alanine aminotransferase(ALT), aspartate aminotransferase (AST) were detected by lactate dehydrogenation method.The ALT and AST kits were from Beckman Kurt Experimental System (Suzhou) Co., Ltd., with batch numbers of AUZ9248 and AUZ8788 respectively; Platelet (PLT) was detected by solid phase coagulation method (batch number: A1042,Hison Meikang Co., Ltd.).All indicators were determined in strict accordance with the operation steps provided in the kit instructions.(3) Noninvasive serum index score: APRI calculation formula:(AST/upper limit of normal value) × 100/PLT[14]; FIB-4 index calculation formula: (age × AST)/(PLT × )[15](4) Adverse reactions and safety evaluation: monitor whether there is secondary damage to liver function (such as ALT, AST), renal function damage (such as creatinine Cr, urea nitrogen BUN), skin itching and other allergic symptoms during treatment.

2.7 Statistical methods

All data were analyzed with SPSS 23.0 statistical software,and the measurement data were expressed with mean ± standard deviation (±s ); Independent sample t-test was used for comparison between groups before and after treatment; Paired t test was used for comparison before and after treatment in the group.The counting data were compared by chi-square test.Pearson correlation analysis was used for correlation analysis.The difference was statistically significant with P＜0.05.

3.Results

3.1 Comparison of serum liver fibrosis index levels between the two groups before and after treatment

There was no significant difference in the levels of HA, LN, PⅢNP and CⅣ between the two groups before treatment, which was comparable (P＞0.05).Compared with the group before treatment,the levels of HA, LN, PⅢNP and CⅣ in the treatment group and the control group decreased significantly (P＜0.05); Compared with the control group, the HA, LN and PⅢNP in the treatment group decreased more significantly after treatment (P＜0.05), while the CⅣlevel did not change significantly (P＞0.05).(See Table 1)

Tab 1 Comparison of four indexes of liver fiber between the two groups before and after treatment(±s ,n=40)

Tab 1 Comparison of four indexes of liver fiber between the two groups before and after treatment(±s ,n=40)

Note: Compared with the group before treatment *) P＜0.05, **)P＜0.01 ;Compared with the control group after treatment, #) P＜0.05.

group time HA(μg /mL) PIIINP(μg /mL) CIV(μg /mL) LN(μg /mL)control group Before treatment 164.31±98.71 27.99±16.36 107.37±68.69 154.72±51.79 After treatment 116.2±69.45*) 17.60±10.59**) 75.30±54.44**) 115.8±32.55**)Treatment group Before treatment 166.80±62.09 28.50±18.83 106.89±74.26 154.68±56.06 After treatment 90.72±37.10**)#) 12.91±6.03**)#) 64.64±44.87*) 98.31±26.38**)#)

3.2 Changes of SWV, APRI and FIB-4 scores of patients in the two groups before and after treatment

Before treatment, there was no significant difference in the scores of SWV, APRI and FIB-4 between the two groups, which was comparable (P＞0.05); After treatment, the scores of SWV,APRI and FIB-4 in the two groups were significantly decreased(P＜0.01).Compared with the control group, the FIB-4 score in the treatment group decreased significantly (P＜0.05), and the SWV and APRI decreased more significantly (P＜0.01), both of which were statistically significant.(See Table 2)

Tab2 Comparison of swv value and non-invasive serum model (APRI score and FIB-4 index) between the two groups before and after treatment(±s ,n=40)

Tab2 Comparison of swv value and non-invasive serum model (APRI score and FIB-4 index) between the two groups before and after treatment(±s ,n=40)

Note: Compared with the group before treatment **)P＜0.01; Compared with the control group after treatment, #) P＜0.05, ##) P＜0.01.

group time SWV APRI FIB-4 control group Before treatment 1.77±1.50 1.76±0.48 2.38±0.48 After treatment 1.48±1.62**) 0.99±0.29**) 1.75±0.53**)Treatment group Before treatment 1.79±1.87 1.71±0.69 2.39±0.89 After treatment 1.29±0.98**)##) 0.59±0.20**)##) 1.20±0.35**)#)

3.3 Study on the correlation between SWV and serum HA, CⅣ, LN, P Ⅲ NP, APRI, FIB-4

Pearson correlation test analysis showed that SWV was positively correlated with FIB-4 (r=0.83), APRI (r=0.82), HA (r=0.87), C Ⅳ(r=0.71), LN (r=0.85), PⅢNP (r=0.77) (see Table 3).SWV is highly correlated with HA, LN, APRI and FIB-4, while SWV is moderately correlated with CIV and PⅢNP.(See Figure 1-2)

Tab 3 Correlation between SWV and serum HA, CIV, LN, PⅢNP and noninvasive serum model APRI, FIB-4

Fig 1 Correlation Analysis of SWV with HA, CIV, LN and PIIINP

Fig 2 Correlation Analysis of SWV, APRI and FIB-4

3.4 Safety indicators

There was no significant difference in the levels of ALT, AST,BUN and Cr between the two groups before treatment, which was comparable (P＞0.05).Compared with the group before treatment,the levels of ALT and AST in the treatment group and the control group decreased significantly (P＜0.01); Compared with the control group, ALT and AST in the treatment group decreased more significantly after treatment (P＜0.05), while the levels of BUN and Cr did not change significantly (P＞0.05).(See Table 5)

3.5 Drug safety evaluation

All the patients who were enrolled in the group and took GDT did not have obvious adverse reactions after treatment, and there was no aggravation of liver and kidney function or new injury.One patient had slight skin itching, which was relieved without special treatment.

Tab 5 Comparison of ALT, AST, BUN and Cr values between the two groups before and after treatment (±s ,n=40)

Tab 5 Comparison of ALT, AST, BUN and Cr values between the two groups before and after treatment (±s ,n=40)

Note: Compared with the group before treatment *) P＜0.01; Compared with the control group after treatment, #) P＜0.01.

group time ALT(U·L-1) AST(U·L-1) BUN(mmol·L-1) Cr(umol·L-1)control group Before treatment 79.52±13.22 69.18±17.05 5.22±1.26 71.02±11.21 After treatment 48.63±10.60*) 48.90±13.19*) 4.86±1.12 71.20±13.27 Treatment group Before treatment 77.45±15.91 70.02±14.87 5.04±1.30 75.37±12.44 After treatment 36.53±8.60*)#) 37.31±7.36*)#) 5.05±0.98 71.84±11.21

4.Discussion

WD is not clearly recorded in ancient Chinese medicine books.According to its clinical manifestations, it is classified as “liver wind”, “tremor” and other categories.The Yellow Emperor’s Internal Classic says: “All the thrusts are fire”, “all the spasms are strong, all belong to damp”.This disease is mainly caused by the deficiency of congenital endowment, the endogenous copper poison,the generation of damp-heat, the accumulation of damp-heat copper poison in the body, and the stagnation of meridians[16].

GDT is an empirical formula obtained from years of clinical observation and summary in the treatment of WD patients with damp-heat accumulation in the Brain Disease Center of my hospital.It is based on the principle of clearing heat and detoxicating, clearing the internal organs and removing dampness, and is composed of scutellaria baicalensis, rhubarb, coptis chinensis, scutellaria barbata,andrographis paniculata, and bixie.Wang Haoze et al.[17-19] showed that emodin could inhibit MAPK pathway and EMT, regulate ERK/Nr77 to promote Gls1 promoter methylation, up-regulate Smad7 and down-regulate-SMA protein expression,so as to play the role of antiliver fibrosis.Liu Shijie et al.[20-22] showed that baicalin can inhibit rat transforming growth factor β 1 expression can play an antihepatic fibrosis role, and can also play an anti-hepatic fibrosis role by inhibiting the expression of related cytokines.Liu Guofang[23]and others found that baicalin can reduce the level of SOCS1 in rats with liver fibrosis, inhibit the proliferation of HSC-T6 cells, and thus alleviate liver fibrosis.The experimental research of Ouyitao et al.[24]shows that berberine can effectively resist liver fibrosis by its antiinflammatory and antioxidant stress effects.The active ingredients such as flavonoids and diterpenoids in Scutellaria barbata have many pharmacological effects such as heat clearing, anti-inflammatory,liver protection and antioxidation[25].Xie Jing et al.[26-27] pointed out that dehydroandrographolide can down-regulate the expression of oxidative stress protein, reduce the activation of hepatic stellate cells, and play an anti-fibrosis role.Other studies have shown that[28-29], GDT can reduce the pathological damage process of WD liver fibrosis by regulating the expression of ERK pathway and MAPK pathway related proteins that cause programmed cell death(PCD), thereby promoting cell copper excretion, and also activating liver Wnt/β- Catenin signal pathway can promote the compensatory self-healing of injured liver tissue and reduce the damage of copper to liver.

The liver is the earliest and most important organ affected by WD[30], and liver fibrosis is the most basic liver pathological change caused by copper metabolism disorder[31].When a large amount of copper ions accumulate in the hepatocytes, the hepatocytes will degenerate and necrosis, the liver will be damaged to different degrees, and the hardness of the liver will gradually change,from liver fibrosis to cirrhosis and liver cancer.Liver fibrosis is a reversible stage, which can be reversed by treatment, but cirrhosis is often difficult to reverse.Therefore, early and effective diagnosis and treatment is of great significance for reversing liver fibrosis[32].For the diagnosis of liver fibrosis, compared with the previous invasive and easily infected liver biopsy, the non-invasive diagnosis method of liver fibrosis has become the first choice for clinical diagnosis in recent years because of its advantages of non-invasive, non-infectious,high accuracy and repeatability[31].The working principle of ARFI is to use the ultrasonic beam to generate the acoustic shear wave signal in the liver tissue, make it locally produce small deformation, and monitor the propagation speed of the low-frequency shear wave to obtain the elastic distribution of the liver tissue[33].Previous studies[34] show that in the diagnosis of liver fibrosis, ARFI, as a reliable means to diagnose the degree of liver fibrosis, has a lower failure rate, and can be used in patients with obesity and ascites, effectively avoiding intrahepatic vessels and ribs, and can quantitatively analyze the degree of liver steatosis.During clinical treatment, ARFI technology is used to observe and quantitatively analyze the degree of liver fibrosis in WD patients at each stage of treatment, which can effectively monitor the condition, clinical efficacy and prognosis of WD patients.In addition, APRI and FIB-4 also have high clinical application value in evaluating the degree of liver fibrosis[35-36], and when combined with the four items of liver fibrosis and ARFI, it can improve the diagnostic rate of liver fibrosis and cirrhosis[7-8].Zhang Jieyuan et al.[37] analyzed the correlation between SWV value and liver fibrosis index in 102 children with biliary atresia, and found that the SWV value of BA children was significantly positively correlated with HA, C Ⅳ, LN, which was the same as the results of this experiment.It shows that SWV can reflect the degree of liver fibrosis together with serum indicators of liver fibrosis, and the higher the shear wave velocity value, the more serious the degree of liver fibrosis.In the results of this study, the SWV value and HA,LN, P Ⅲ NP in the treatment group were significantly lower than those in the control group, indicating that GDT can significantly improve liver fibrosis and improve the quality of life of patients.

To sum up, GDT combined with DMPS can significantly improve the degree of liver fibrosis in WD patients.As a traditional Chinese medicine decoction, GDT has the advantages of small adverse reactions, high safety and low price.It is worth promoting as a clinical treatment plan for patients with liver fibrosis due to dampheat accumulation.The inadequacies of this study are that the sample data is small, the capacity is limited, and the research time is relatively short.There may be statistical errors in statistical analysis.Therefore, large sample, random, long-term research will be carried out later to reduce statistical errors and provide more data support.This paper focuses on liver research, but WD can affect kidney and heart, etc.In the later stage, our research group will further carry out kidney and heart research.

Statement of conflicting interests of the author

All authors have no conflict of interest.

Author’s contribution

Wang Meixia: design research ideas, fund support and paper review; Han Hui: Scheme implementation and thesis writing; Tao Zhuang, Zhang Yanyun: paper revision, document retrieval; Zhang Yu: data review; Jiang Zhenzhen: Data collection; Wang Fei, Sun Kuo: Statistical analysis.