Efficacy of Traditional Chinese Medicine on Animal Model of IgA Nephropathy: A Systematic Review and Meta-Analysis

Objective Traditional Chinese medicine (TCM) has a long history in the treatment of Immunoglobulin A nephropathy (IgAN). A large number of animal experiments focused on the TCM treatment of IgAN are conducted every year. The evidence for these preclinical studies is not clear. This study summarized and evaluated the results of animal experiments on TCM treatment for IgAN. Methods We systematically searched animal studies from 6 databases from inception to August 30, 2022. We included Chinese studies from the key magazine of China technology. The quality of the included studies was evaluated with the SYRCLE animal experimental bias risk assessment tool and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Results Out of 832 records identified in the initial search, 30 studies were selected. The results indicated that, compared with the control group, the TCM treatment group improved 24 h urine protein (24 h-UP) level (standardized mean difference (SMD) 3.57, 95% confidence interval (CI) 4.48 to 2.66, P < 0.001), urine red blood cell (U-RBC) (SMD 13.66, 95% CI 17.99 to 9.32, P < 0.001), serum creatinine (Scr) (mean difference (MD) 10.89, 95% CI 17.00 to 4.77, P < 0.001), blood urea nitrogen (BUN) (MD 2.44, 95% CI 3.42 to 1.47, P < 0.001), tumor necrosis factor-α (TNF-α) (MD 171.28 to 95% CI 323.68 to 18.88, P=0.03), transforming growth factor-β1 (TGF-β) (SMD 4.02, 95% CI 7.26 to 0.77, P=0.02), matrix metalloproteinase-9/tissue inhibitors of metalloproteinase-1(MMP-9/TIMP-1) (MD 0.03, 95% CI 0.00 to 0.06, P=0.02), nephrin mRNA (SMD 3.39, 95% CI 2.59 to 4.18, P < 0.001). However, there is no difference in albumin level (MD 1.10, 95% CI 0.06 to 2.26, P=0.06) and interleukin-6 (IL-6) (MD 170.77, 95% CI 365.3 to 23.75, P=0.09). Conclusions TCM can improve 24 h-UP, U-RBC, Scr, BUN, MMP-9/TIMP-1, TNF-α, TGF-β, and nephrin mRNA of IgAN animal models. Moreover, there is a need for rigorous reporting of preclinical research methodology, which is essential to support the quality of preclinical research. Registration. This review was registered with a systematic review record CRD42020171404 in the PROSPERO database.


Introduction
IgA nephropathy (IgAN) refers to primary glomerulonephritis with a large number of IgA or IgA-based immune complex granules deposited in the mesangial area. It is the most common primary glomerulonephritis in the world at present. [1] Te modern medical treatment for IgAN is limited [2,3]; Chinese medicine (including herb decoctions under the guidance of TCM theory, extract of herbs or decoctions, and patents) has been widely approved for its positive role in the prevention and treatment of IgAN [4].
Te incidence of primary glomerular disease accounts for 30% in Asia, and it is also one of the leading causes of end-stage renal disease (ESRD) in China. Data show that the incidence of this disease has been gradually increasing in recent years. [5] Te prognosis of IgAN is not optimistic. After treatment, about 50% of patients still progress to ESRD within 30 years. [6] Patients with ESRD can only rely on kidney transplantation or dialysis to maintain their lives, which puts a massive psychological and economic burden on individuals, families, and society. Kidney function preservation and remission of proteinuria are the principles for its treatment. [7] Glucocorticoids and immunosuppressants can reduce urinary protein levels in IgAN patients and improve the prognosis. [3] However, severe adverse reactions, insensitivity, and drug resistance occur sometimes. TCM has certain advantages in the treatment of IgAN. It could delay the development of IgAN in the early stage, synergistically treat IgAN with immunosuppressants and glucocorticoids in the middle stage, and improve life quality in the end stage. [8] However, the results of these studies were not systemically evaluated. Systematic evaluation of animal experiments has become a new trend and an essential means to integrate animal experimental results, improve the quality of animal experiments, and guide clinical research. It is also an important channel to connect basic research and clinical trials. To further clarify the mechanism of Chinese medicine in the treatment of IgAN, this study intends to use the meta-analysis method to systematically evaluate the intervention of TCM in experimental IgAN animal models, to expand further the evidence of the mechanism of TCM in the treatment of IgAN, and to provide reference data for follow-up research.

Materials and Methods
Tis review was registered with a systematic review record CRD42020171404 in the PROSPERO database.

Search Strategy.
Databases including PubMed, Cochrane Library, Embase, CNKI, VIP, and Wanfang data were searched for the literature from the inception to 4 April 2020 without language restriction. MeSH terms and keywords ("Immunoglobulin A Nephropathy" OR "Immunoglobulin A") AND ("Chinese Medicine" OR "Chinese Herbal Medicine") AND ("Animal" OR "Rat" OR "Mice") were used to search studies.

Exclusion Criteria.
(1) Participants: in vitro studies and research in humans; (2) intervention: TCM without dose unit or TCM was not given by oral gavage administration; (3) control: other TCM; (4) study design: case reports, crossover studies, and studies without a separate control group; (5) pilot studies; (6) reviews; (7) conference papers; (8) studies without full text. If studies were repeatedly published, we chose the one with the largest sample size.

Data Extraction.
Two reviewers (TYC and HAW) independently extracted the following information according to the predesigned fle from selected studies: characteristics of studies (frst author, publication year, TCM treatment composition, type of models, modeling method; individual data for each study, including animal number, species, gender, weight, treatment time, and mode of administration). Te outcome measurement includes 24 h-UP, U-RBC, Scr, BUN, Alb, MMP-9/TIMP-1, IL-6, TNF-α, TGF-β, and nephrin mRNA. If outcomes were presented at diferent time points, variables were extracted from the last time point. If studies had more than one experiment group sharing one control group, the control group would be separated into multiple groups (the number was the same as the experiment groups), incorporating these comparisons into this meta-analysis. Any disagreements regarding extracted data were resolved through discussion, if necessary, by a third party (SLZ).

Statistical Analysis.
We used RevMan version 5.3 by the Cochrane Collaboration Network to analyze the data. Continuous data were expressed as the mean diference with a 95% confdence interval (CI). Categorical data were calculated with the risk ratio (RR) and 95% CI. We evaluated heterogeneity with the Higgins I 2 test. If I 2 > 50%, a randomefect model was used for meta-analyses; if not, a fxed-efect model was used. Publication bias was assessed by a funnel plot. Te quality of evidence was rated with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) [9].

Study Inclusion.
In the initial search, 832 related studies were obtained and 206 repetitive studies were excluded as duplications by EndNote X9 software. Te rest studies were further screened by reviewing titles, abstracts, and the list of Chinese Core Journals. Te rest 52 studies were full-text screened, and 19 studies were excluded by a nonunifed treatment dose. At last, 30 eligible studies were identifed. All the literature was published in Chinese, and all trials were conducted in China. Te literature screening process and results are shown in Figure 1.

Publication Bias Test.
We conducted a funnel plot to indicate publication bias and potential publication bias observed in Figure 13.
3.16. Sensitivity Analysis. Signifcant heterogeneity was found in the meta-analysis of 24 h-UP, U-RBC, Scr, BUN, albumin, IL-6, TNF-α, and TGF-β1. We conducted a series of sensitivity tests by excluding better-designed studies to examine the robustness of our results, and we found that the results were consistent.

Discussion
Te renal protective efect of TCM has been extensively researched in diferent animal experimental models, and our systematic review and meta-analysis intend to analyze whether TCM treatment exerted an efect on IgAN animal models. Te results indicated that oral gavage TCM could signifcantly lower levels of 24 h-UP, U-RBC, Scr, BUN, albumin, MMP-9/TIMP-1, IL-6, TNF-α, TGF-β1, and nephrin mRNA. Twenty-one TCM treatments were used in our systematic review, eleven were decoctions, and eight were patent. Te main medicinal ingredients of TCM are the following: Rehmannia, Astragalus, Chinese yam, Puhuang,    [42] Consequent immune complexes deposit in the glomerular mesangium, which activates the complement pathway, stimulates mesangial cells, and induces the secretion of cytokines, fnally resulting in infammation and fbrosis. IgAN is an autoimmune disease wherein immune complexes cause renal injury. [43] Studies of animal experiments showed that urinary protein could induce renal tubular epithelial cell damage, so urinary protein has been used as an independent factor in evaluating renal prognosis. [44,45].
Te pathogenesis of IgAN includes environmental factors, genetic factors, and immune factors, among which immune factors have been the primary targets for the study of the treatment of the disease. At present, the cytokines that play an essential role in the pathogenesis and progression of IgAN are IL-6, TNF-α, TGF-β1, and MMP-9/TIMP-1. TNFα is produced by activated monocytes; under normal conditions, an appropriate amount of TNF-α has a protective efect on the body, and excessive TNF-α causes immune damage to the body. Studies have shown that mononuclear macrophages infltrate into renal tissue to release TNF-α, resulting in focal glomerular damage, resulting in gross hematuria, [46,47] consistent with clinical studies. [47,48] IL-6 can induce B lymphocytes to diferentiate and produce immunoglobulin, stimulate the proliferation of glomerular mesangial cells, and produce an extracellular matrix, thus increasing the burden on the kidney and leading to glomerular fbrosis. [49] Glomerulosclerosis is a common            MMP-TIMP complex at 1 : 1, blocking the binding of MMP-9 to the substrate, thus afecting the balance of ECM accumulation and degradation. [53,54] Any imbalance between TIMP-1/MMP9 and ECM may lead to abnormal accumulation of ECM, glomerular disease, glomerular remodeling, hematuria, and proteinuria. Te meta-analysis results showed that after TCM treatment, TNF-α and TGF-β could be reduced, and serum MMP-9/TIMP-1 content could be increased; the diference was statistically signifcant. In addition, the study found that there is also podocyte destruction in the occurrence and development of IgAN. [55] Podocyte injury, especially the role of changes in the fssure diaphragm in the occurrence of IgAN proteinuria, has also attracted people's attention. Clinical studies have confrmed that the urinary protein level exceeding 1.0 g/ d during renal biopsy indicates that the prognosis of patients is poor. [56,57] Terefore, stabilizing the podocyte fssure diaphragm will be an important starting point for treating IgAN proteinuria. Nephrin, a sign of mature podocytes, is the frst transmembrane protein found on the glomerular fltration barrier of SD, and it is also the "main body" part of SD. Te nephrin defciency in humans and rats will lead to classic SD defciency and massive proteinuria. Te results of the meta-analysis showed that the TCM treatment could signifcantly reduce the level of urinary protein and downregulate the expression of nephrin mRNA in podocytes in rats with IgA nephropathy, thus playing a role in the treatment of IgAN.
Plenty of animal experiments focused on TCM treatment for IgAN animal models have been conducted for years. For better quality of included studies, we chose studies from the list of Chinese Core Journals if published in Chinese. Animal models for IgAN were established in three kinds of rats/mice. Six studies constructed animal models with Wistar rats, one study constructed an animal model with BALB/C mice, and twenty-three studies used SD rats. Most studies constructed models with two methods. One method is oral gavage with BSA 400 mg/kg every other day, subcutaneous injection of CCL4 0.1 ml/ weekly and benne oil 0.5 mL/weekly for nine weeks, and intravenous injection of LPS 0.05 mg at the sixth or eighth   week. Te other method is intravenous injection of SEB combined with oral gavage with BSA. IgAN animal experiments conducted outside China usually construct an animal model with a ddY mouse [58] which has an abnormally high concentration of IgA from 10 to 60 weeks, but rare hematuria. Te BSA + SEB method usually constructs models with a high mortality rate after modeling. Most included studies used the BSA + LPS + CCL4 method to construct an animal model.
Methodological quality was low among animal experiments. We used GRADE to evaluate the certainty of the main outcomes of this meta-analysis, and all certainty is very low. Insufcient reporting of how these studies were conducted lowers the quality and increases the biases. Six of ten SYRCLE's tool items were reported as unclear in all included studies, which means researchers need more training in methodological ability. Evidence-based medicine ability should be emphasized to TCM researchers and practitioners. Te animal modeling method is also a source of heterogeneity. Furthermore, our results were consistent with the sensitivity of excluding studies with diferent modeling methods.
Trough this study, we can fnd that although there are a large number of IgAN animal experimental studies published, most of them are not fully reported in the methods of model establishment, animal feeding, and model evaluation. High-quality systematic evaluation of animal experiments will help to prevent the waste of laboratory animals and test participants by carrying out unnecessary, inefective, or less information research [59]. Terefore, a high-quality original research design is essential to provide research evidence for treating IgAN with traditional Chinese medicine.
Tis study showed that TCM intervention in IgAN animals could reduce levels of 24 h-UP, U-RBC, Scr, BUN, TNF-α and upregulate the contents of serum TGF-β1, nephrin mRNA, and MMP-9/TIMP-1. According to different animal species and modeling methods in the subgroup analysis, the results show that TCM signifcantly afects IgAN SD and Wistar rats; the results are statistically signifcant. Tis meta-analysis reinforces the evidence that TCM has a protective efect in experimental IgAN animal models. However, the methodological quality of these studies needs to be improved.

Data Availability
Te datasets used and/or analyzed during the present study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.