Treatment of Liver Fibrosis after Hepatitis B with TCM Combined with NAs Evaluated by Noninvasive Diagnostic Methods: A Retrospective Study

Objective. Chronic hepatitis B liver fibrosis is an important intermediate link in the development of liver cirrhosis. A retrospective cohort study was conducted in Longhua Hospital affiliated to the Shanghai University of Traditional Chinese Medicine in order to prove whether integrated traditional Chinese and Western medicine could improve the incidence of CHB complications and clinical prognosis. There are 130 patients with hepatitis B liver fibrosis (being treated from 2011–2021) included in the study, and the patients were divided into 64 TCM users (NAs combined with TCM) and 66 TCM nonusers (NAs antiviral therapy). The serum noninvasive diagnostic model (APRI, FIB-4) and LSM value were used to classify the stages of fibrosis. The results showed that the LSM value was decreased significantly in TCM users compared with TCM nonusers (40.63% versus 28.79%). Indicators of FIB-4 and APRI of TCM users have improved significantly compared with that of TCM nonusers (32.81% versus 10.61% and 35.94% versus 24.24%). The AST, TBIL, and HBsAg levels in TCM users were lower than those in TCM nonusers, and the HBsAg level was inversely correlated with the CD3+, CD4+, and CD8+ in TCM users. The PLT and spleen thickness of TCM users also were improved considerably. The incidence rate of end-point events (decompensated cirrhosis/liver cancer) in TCM nonusers was higher than that of TCM users (16.67% versus 1.56%). The long course of the disease and a family history of hepatitis B were the risk factors for disease progression, and long-term oral administration of TCM was the protective factor. As a result, the serum noninvasive fibrosis index and imaging parameters in TCM users were lower than those of TCM nonusers. Patients in the treatment of NAs combined with TCM had better prognoses such as a lower HBsAg level, a more stable lymphocyte function, and a lower incidence of end-point events. The present findings suggest the effect of TCM combined with NAs in the treatment of chronic hepatitis B liver fibrosis is better than that of single drug treatment.


Introduction
Chronic hepatitis B liver fbrosis (hepatitis B liver fbrosis for short) is an abnormal change in the liver structure and/or function caused by long-term chronic stimulation of the hepatitis B virus and excessive proliferation and deposition of extracellular matrix (ECM) components. It is an important intermediate link in the development of liver cirrhosis [1]. More than 2 billion people worldwide are infected with HBV, of which 240 million people develop chronic reversing liver fbrosis is only 30-40%, and fbrosis may still exist and continue to develop after viral response [4][5][6]. Liver fbrosis is one of the major global health problems due to the lack of efective treatment. As a reversible stage in the development of chronic hepatitis B (CHB), liver fbrosis plays an important role in preventing the occurrence and development of liver cirrhosis and prolonging the survival of patients with chronic liver disease.
Te efect of traditional Chinese medicine (TCM) combined with nucleotide analogues (NAs) in the treatment of chronic hepatitis B liver fbrosis is better than that of single drug treatment in some studies [7]. As the main method to prevent and treat the progress of liver fbrosis, integrated traditional Chinese and Western medicine can improve the incidence of CHB complications and clinical prognosis. However, there is a lack of long-term follow-up of patients with liver fbrosis. Terefore, this study aims to determine the impact of long-term TCM syndrome diferentiation combined with NAs antiviral therapy on the clinical efcacy and prognosis of patients with chronic hepatitis B liver fbrosis.

Study Subjects.
A retrospective cohort study was conducted using inpatient and outpatient data sets from 2011 to 2022 in the department of infectious diseases, Longhua Hospital. Te data used for analysis include data from the electronic medical record (EMR) and laboratory information systems (LIS), as well as detailed procedures, medication, and TCM prescriptions based on EMR. As shown in Figure 1, taking "liver fbrosis after hepatitis B" as the keyword, a total of 406 patients were screened out, 276 patients of which were excluded from the study.
Te diagnostic criteria of the noninvasive liver fbrosis refer to the 2019 guidelines for the diagnosis and treatment of liver fbrosis with integrated traditional Chinese and Western medicine [8], and the inclusion criteria are as follows: (1) Treatment time with NAs ≥1 year, if the recipient of traditional Chinese medicine has been taking it for at least a year. (2) Te liver stifness measurement (LSM, FibroScan) was ≥7.0 kPa [9,10], abnormal elevation of the serum noninvasive diagnostic model: ARPI ≥ 1 or FIB-4 ≥ 1.45 [11,12]. (3) TCM diagnosis conforms to the diagnostic criteria for liver fbrosis formulated by the Liver Disease Professional Committee of the Chinese Association of Integrative Medicine [7]. (4) Te patients were followed up in Longhua hospital for ≥5 years, and the relevant laboratory indicators of the patients were measured at least once a year during the follow-up period. (5) Major demographic and clinical data are available, such as LSM. Tere were 130 patients meeting the inclusion criteria fnally.
When patients who received TCM for more than 3 months each year were regarded as TCM users, patients who met the inclusion criteria were divided into two groups (TCM users and TCM nonusers), and there are 64 TCM users and 66 TCM nonusers. Te details are as follows: TCM nonusers: NAs monotherapy: entecavir tablets (ETV) 0.5 mg/day or tenofovir disoproxil (TDF) 300 mg/day or tenofovir alafenamide fumarate tablets (TAF) 25 mg/day or lamivudine (LAM) 100 mg/day or adefovir dipivoxil Tablets (ADF) 10 mg/day or LAM 100 mg coupled with ADF10 mg/ day. NAs are taken orally every day without interruption.
TCM users: oral NAs combined with TCM. Individualized TCM prescriptions can be taken discontinuously by patients according to the analysis of TCM syndrome distinction, but the total amount of time receiving TCM annually must be more than three months.

Efcacy Evaluation.
Te main outcome measures were the infuence of TCM treatment on liver fbrosis-related indexes, as well as the impact on the occurrence of liver cirrhosis decompensation and/or liver cancer. According to the evaluation standard of the serum noninvasive diagnostic model, the categorization is carried out following with the values of APRI, FIB-4, and LSM. Obvious fbrosis (F2) is defned as 1 ≥ ARPI < 2 or 1.45 ≥ FIB-4 < 3.25 or 7 ≥ LSM < 9.0 kPa; severe fbrosis (F3)/cirrhosis is defned as ARPI ≥ 2 or FIB-4 ≥ 3.25 or LSM ≥ 9.0 kPa. Grading of the data both before and after the treatment, the symptom was improved if the grade in fbrosis decreased. Te symptom was steady if there was no change in grade. If the grade increased compared with the prior grade, it meant symptom progression. We compared the changes of HBsAg, HBeAg, and lymphocyte subpopulation after treatment between the two groups to understand the efect of viral immunity on liver fbrosis. Te ending occurrences were decided based on the Child's Pugh grade, tumor marker AFP, liver contrastenhanced CT, or MRI.

Safety Index.
Alanine aminotransferase (ALT) and serum creatinine (Scr) were used to evaluate the safety.

Statistical
Analyses. Statistical data were described as mean ± standard deviation, median, or percentage. Diferences in baseline characteristics between the two groups were assessed by Student's t-test or the chi-squared test. If the measurement data obeyed the normal distribution, it was expressed as mean ± standard deviation ( ± s). Te paired ttest was used to compare the data within the group before and after treatment, and the independent sample t-test was used to compare the data between the groups. If the normal distribution was not obeyed, the data was expressed by the median (interquartile interval) [M (p25-p75)]. Te Wilcoxon signed rank sum test of paired data was used for the comparison of intragroup data before and after treatment, and the Mann-Whitney U signed rank sum test of two independent samples was used for the comparison of intergroup data. Pearson was used for comparison of grade data X2-test. Te LR positive method of the logistic hazard regression model was used to analyze the independent factors afecting the incidence rate of endind events (decompensated liver cirrhosis or liver cancer). Te relative risk (RR) was used to determine whether the exposure factors were related to results. SPSS v25.0 was used for statistical analysis. P < 0.05 (bilateral) was considered statistically signifcant. Table 1 describes the baseline characteristics of the patients. Tere were 49 males and 15 females in the TCM users. Te average age was (47.67 ± 10.70) years. Te follow-up time ranged from 5 to 15 years, with a median time of 7 years. In the TCM nonusers, there were 41 males and 25 females. Te average age was (52.98 ± 10.14) years. Te time of follow-up ranged from 5 to 11 years, with a median time of 8 years. Tere were no statistical diferences in baseline characteristics between the two groups, including age, gender, course of disease, smoking history, drinking history, family history, ALT level, HBeAg level, and viral load.

Comparison of Liver Function Indexes between the Two
Groups. Te Table 2 indicates that AST and TBIL in the TCM users were signifcantly improved compared with those in the TCM nonusers.
Te date analysis of repeated measurements was performed when we ensured that the data of all patients during the follow-up period met the normality and homogeneity of variance. Te dynamic observation of the liver function was managed to perform on 130 patients who were followed for 5 years as shown in Figure 2. It was found that the levels of ALT and AST in the two groups were lower than those before treatment, and the diference of AST between the two groups was statistically signifcant. Tere were no statistical diferences in ALB and TBIL after treatment compared with before treatment.
According to the results of the time-dependent efect test represented by the follow-up time, there were statistical diferences in the levels of ALT and AST at diferent times (P Time < 0.001). Te results of the intergroup efect test showed that there were statistical diferences in the AST levels between TCM users and TCM nonusers (P Group � 0.006). Tere was no statistical diference between ALB and TBIL in the test of the time efect and intergroup efect.

Comparison of Noninvasive Liver Fibrosis Indexes between
the Two Groups before and after Treatment. Figure 3 compares the changes of FIB-4, APRI, and LSM between the two groups after treatment. Calculating the two groups' fbrosis grading data, fbrosis was improved in the 40.63% TCM users and 28.79% TCM nonusers according to the LSM (P � 0.038). Fibrosis was improved in the 32.81% TCM users and 10.61% TCM nonusers in FIB-4(P � 0.009). Fibrosis was improved in the 35.94% TCM users and 24.24% TCM nonusers in APRI (P � 0.039).
As shown in Table 3, 37 TCM users and 45 TCM nonusers were compared before and after the treatment fnally by excluding the patients with missing four items of the liver fber during the follow-up period. Because the four items of the liver fber did not obey the normal distribution,  Evidence-Based Complementary and Alternative Medicine the median (the interquartile interval) was used to represent the baseline. Although there were diferences in the median between the two groups, especially the HA level (the normal range of 0-100 ng/ml), there was no statistical diference between the two groups in the baseline characteristics of the four items of liver fber according to the rank sum test. Compared to the TCM nonusers, the hyaluronic acid (HA) and collagen type IV (CIV) of TCM users were considerably improved after the treatment. HA decreased signifcantly from the second year to the ffth year after treatment, and CIV considerably declined between the fourth and ffth years following treatment. Compared with before treatment, the four items of the liver fber were considerably reduced after TCM treatment. Te levels of laminin (LN) and CIV decreased signifcantly after treatment in TCM nonusers, whereas other items did not change appreciably.

Viral Immunity and Lymphocyte Immune Function.
After excluding the patients with incomplete HBV markers during the follow-up, there were 61 patients in the TCM group and 62 patients in the non-TCM group. We observed      (Table 4). When HBsAg level ≥ 250, the level was marked as 1, and HbsAg level < 250, the level was marked as 2. According to the grading changes before and after treatment, it could be divided into improvement, stability, and progress. Te results showed that the level of HBsAg in the TCM group was signifcantly decreased (P � 0.019), indicating that TCM treatment has a certain efect on reducing the HBsAg level. We marked HBeAg negative as 1 and HBeAg positive as 2, and the HBeAg level in the two groups was observed after treatment. Te results showed that there was no signifcant diference between the two groups (P � 0.759). Because the sample of HBeAg-positive patients in the 130 patients was too small, the HBeAg negative conversion rate and serum conversion rate were not counted in this study. As shown in Table 5, the relative counts of CD3+, CD4 +, and CD8+ cells in the TCM users were statistically diferent compared to those in the TCM nonusers, and CD16+ increased signifcantly compared to that before treatment. Te lymphocyte subpopulation decreased after treatment in the TCM nonusers, and CD3+, CD4+, and CD8+ levels statistically decreased compared with those before treatment.
As shown in Figure 4, low levels of serum HBsAg were correlated with elevated lymphocyte levels, particularly CD3+, CD4+, and CD8+. Additionally, the high level of CD8+ was signifcantly correlated with a low level of LSM.

Comparison of PLT, Spleen Tickness, and Length between
the Two Groups before and after Treatment. Patients with incomplete B-ultrasound data during follow-up were excluded (6 cases in each group). Te changes in the spleen and platelet (PLT) between the two groups after treatment are shown in Table 6. Te value of PLT in both groups was higher than before treatment, in which the PLT level and spleen thickness of the TCM users were signifcantly changed compared with those of the TCM nonusers. Meanwhile, the thickness and length of spleen increased signifcantly after treatment in the TCM nonusers. According to the long-term follow-up observation of the PLT level in Figure 5, the PLT level steadily increased with the prolonging of follow-up time. Table 2, there were no signifcant adverse events, and the ALT of both the groups remained normal during the treatment. Te Scr level had no signifcant diference between the two groups after treatment (Table 7).

Prognosis and Risk.
During the follow-up period, there were 12 patients (1 TCM user and 11 nonusers) who sufered liver cancer and/or cirrhosis decompensation. Accordingly, the incidence of ending events increased by 16.67% in the TCM nonusers (compared with 1.56% in the TCM users). Te distribution of ending (decompensated cirrhosis and/or liver cancer) in each group is shown in Figure 6. It is noteworthy that there was only one liver cancer patient in the TCM users. As indicated in Table 8, the potential risk factors for the ending were identifed and logistic regression analysis was conducted to investigate the risk factors for the ending. Te results showed that the P value of variable X1, X10, and X11 was less than 0.05, and the diference was statistically signifcant, suggesting that the course of the disease, family history, and whether to use TCM were the relevant risk factors for the occurrence of ending. According to whether TCM is used as the exposure factor, when the TCM user is in the exposure group, results of the relative risk analysis showed that RR = 0.079 (Table 9), indicating that TCM is benefcial exposure. On the contrary, the RR of disease's course and family history were 9.603 and 21.600, respectively, which were risk factors. , and Duanlonggu (Os Draconis). Tese medicines have the function of softening hardness and dissipating mass, spleen fortifying, and calming/soothing the liver, which demonstrate that "soften hardness and disperse knots" and "soothe the liver and fortify the spleen" are the main treatment methods of TCM for liver fbrosis after CHB.    Note. Compared with that before treatment in this group, * P < 0. 05; compared with the control group after treatment, △ P < 0. 05.

Discussion
Te occurrence and development of fbrosis caused by chronic viral hepatitis B are afected by viral factors (such as viral genotype and gene variation, viral load, and viral antigen), host factors (such as immune response, immune escape, cytokines, complement components, and human leukocyte antigen genotype), and their interactions [13]. Hepatitis B virus induces immunosuppressive cells, such as MDSCs, NK-reg, and T-reg cells through an immunosuppressive cascade [14][15][16]. Excessive immunosuppression may lead to persistent HBV infection, congenital and adaptive immune response dysfunction, liver fbrosis, and HCC progression.
Many clinical studies have shown that TCM has signifcant advantages in blocking-up liver fbrosis, controlling the development of liver infammation, and improving clinical symptoms [17]. LSM is accepted as a highly  Note. Compared with that before treatment in this group, * P < 0 05; compared with the control group after treatment, △ P < 0.05.  Evidence-Based Complementary and Alternative Medicine reproducible and accurate indicator for the assessment of liver fbrosis. Terefore, FibroScan was used to track the improvement in liver stifness brought on by TCM, and the results showed that the rate of improvement after treatment was 40.63%, which was much better than the 28.79% of the TCM nonusers. APRI and FIB-4 are relatively mature serum noninvasive detection models, in which the sensitivity and specifcity of APRI in evaluating liver fbrosis after hepatitis B are 0.87 and 0.66 [18] and of FIB-4 are 0.78 and 0.65 [19], respectively. According to results of our research, both of them efectively predicted the improvement of TCM on liver fbrosis. By analyzing the four items of liver fbrosis, we found that HA and CIV in patients receiving TCM together with NAs were statistically considerably better than the TCM nonusers. Existing research studies have found that elevated serum Col IV is related to degrees of fbrosis and severity of hepatitis, and it is served as a histochemical marker of perisinusoidal basement membrane formation in liver disease [20]. Most researchers have proved that there is a good correlation between serum hyaluronic acid levels and liver fbrosis development [21], and it can be considered as a reliable noninvasive glycoprotein for discriminating the patients with liver fbrosis from the healthy ones to assess the liver fbrosis grade [22].
Te AST level is one of the independent predictors of signifcant fbrosis [11], which was used in the liver fbrosis noninvasive diagnostic model of APRI and FIB-4. Te serum bilirubin level represents hepatic synthetic and excretory function, which is the component of most well-recognized prognostic models including the -Child-Pugh score and model for end-stage liver disease (MELD) score [23]. Our data shows that AST and TBIL after TCM treatment are signifcantly lower than those in the TCM nonusers, and AST levels are diferent between the diferent groups at each time point of follow-up, suggesting that AST and TBIL may also predict the severity and long-term prognosis of patients with chronic hepatitis B liver fbrosis.
In patients with CHB, HBsAg plays an important role in predicting the level of liver fbrosis in HbeAg positive and HbeAg negative patients [24]. However, when CHB is established, HbsAg serum clearance is rarely observed, which may be due to the impairment of innate and adaptive immunity and the exhaustion of T-cell and B-cell responses [25]. Te activation of antiviral T cells contributes to the decline of HbsAg [24]. Our results showed that the majority of TCM users' serum HbsAg levels decreased after treatment, while the increase of lymphocyte CD3+, CD4+, and CD8+ was signifcantly correlated with low-level HbsAg. Tese results indicate that TCM combined with antiviral drug NAs can help maintain a low baseline of HbsAg and HBV DNA by improving lymphocyte levels.
An experimental research has suggested that fbrosis induced by acute HBV infection leads to the immune disorder of intrahepatic lymphocytes and the immune function is inhibited [26]. As hepatitis B virus replication enters the chronic phase, it is characterized by quantitatively and qualitatively weak HBV-specifc CD8+ T-cell responses. Te progress of liver fbrosis during HBV infection may also jeopardize hepatocellular antigen recognition by intravascular CD8+ T cells when crawling along liver sinusoids [27]. Meanwhile, the presence of fbrosis/cirrhosis was also associated with a lower B cell allostimulatory capacity, manifested in the reduced ability of alloreactive CD4+ T cells producing the antifbrotic factor after the B-cell contact [28]. Te number of CD3+, CD4+, and CD8+ in patients treated   10 Evidence-Based Complementary and Alternative Medicine with TCM was signifcantly higher than that in TCM nonusers, and there was a negative correlation between CD8+ and the noninvasive fbrosis index (LSM), which indicates that TCM has a defnite impact on restoring and enhancing the lymphocyte immune function and inhibiting fbrosis.

Evidence-Based Complementary and Alternative Medicine
According to the TCM theory, liver fbrosis after CHB is caused by HBV invading the body and being alive for a long time. In addition, improper diet, heavy labor, unsmooth mood, insufcient healthy qi, etc. can damage the immune function. Besides, the virus stays in the liver for a long time, so the liver cannot work normally. Te disease location is mainly related to the liver, and the spleen, stomach, and kidney also are involved [29], which is consistent with our statistical results (Figure 7). In terms of treatment, the consensus believes that liver diseases were caused by "upright weakness" and "blood stasis" [7]. Te prescription data demonstrates that the single herbs with a frequency of more than 50% are mainly softening-hardness and stasiseliminating medicinal such as Ostreae concha, Carapax trionycis, Arcae concha, and Radix Salviae Miltiorrhizae, and spleen-fortifying medicinal such as Atractylodes macrocephala, Glycyrrhiza uralensis fsch, and Poria cocos; the latter helps to improve patients' own immunity and reduce the continuous harm of the virus to the body.
In modern pharmacological research, they mainly play the role of anti-infammatory agents and liver protection, inhibiting the activation of hepatic stellate cells, reducing liver fbrosis, regulating immunity, and enhancing the lymphocyte function. For example, licorice total favonoids, a natural source of hepatoprotective activity, can heal liver tissue and reduce hepatic injury via alleviating infammation, improving the antioxidant enzyme activity, and reducing oxidative stress in liver tissue [30]. Te hepatoprotective efects of Poria cocos polysaccharides are related to the molecular mechanisms of suppressing cell death, reducing hepatocellular infammatory stress, and heat shock protein bioactivity [31]. Both the extract and pure peptides of Carapax trionycis display strong hepatoprotective efects. Among them, oligopeptide I-C-F-6 has been proved to have a antifbrosis efect in the animal model of liver fbrosis, the mechanism of which is related to modulating NF-kB and Wnt/b-catenin signaling [32]. In immunocompromised mice, oyster oligopeptide could signifcantly enhance the proliferation of spleen lymphocytes, NK cell activity, regulate the distribution of CD4+T and CD8+T spleen lymphocyte subsets, and positively regulate the cell immunity, humoral immunity, and nonspecifc immune function [33]. Oyster peptide can restore the index of the thymus, spleen, and liver, stimulate cytokine secretion, and afect the occurrence and duration of chronic hepatitis B by regulating the balance of T1/T2 [34].
Tere are limitations in this study. Firstly, because it is not a randomized and controlled trial, there may be a potential bias due to the infuence of economy, work, and family environment. Some patients choose diferent oral methods of TCM preparations, which will have diferent efcacy, so a further research is needed. Secondly, due to the limitation of detection technology, HBV DNA cannot detect the amount of virus below 100 IU/ml, and it is impossible to

Conclusions
In conclusion, this retrospective study indicates that TCM combined with NAs in the treatment of patients with liver fbrosis after chronic hepatitis B can signifcantly improve fbrosis indicators, stabilize the liver function, maintain a low baseline of HBsAg and HBVDNA, promote the recovery of patients' immune system, and help block or even regress the process of liver fbrosis in patients with chronic hepatitis B.