Celastrol ameliorates lupus by promoting apoptosis of autoimmune T cells and preventing autoimmune response in MRL/lpr mice.

OBJECTIVE: Celastrol is a bioactive constituent extracted from Tripterygium wilfordii (thunder god vine). It has been demonstrated to have a therapeutic effect on experimental disease models for chronic inflammatory and immune disorders. In the present study, we investigated whether and how celastrol exerts a regulatory effect on the autoimmune response in MRL/lpr mice. METHODS: We performed an in vivo study to determine the therapeutic effects of celastrol in MRL/lpr mice and then further investigated the underlying mechanism of celastrol in the regulation of the autoimmune response in MRL/lpr mice. RESULTS: Celastrol showed a therapeutic effect in MRL/lpr mice by preventing the enlargement of the spleen and lymph nodes, alleviating renal injury, and reducing the levels of ANA and anti-double-stranded DNA antibodies. Furthermore, celastrol suppressed the in vivo inflammatory response in MRL/lpr mice by reducing the serum levels of multiple cytokines, including interleukin (IL)-6, tumour necrosis factor (TNF) and interferon (IFN)-γ, and the production of multiple antibody subsets, including total IgG, IgG1 and IgG2b. In vitro, celastrol reduced anti-CD3 antibody stimulation-induced T helper 1 and TNF-producing cells in CD4+ T cells of MRL/lpr mice. In addition, celastrol significantly affected B cell differentiation and prevented the generation of plasma cells from B cells in MRL/lpr mice by reducing the frequency of activated and germinal centre B cells. Celastrol treatment also affected T cell differentiation and significantly reduced central memory T cell frequencies in MRL/lpr mice. Importantly, celastrol treatment specifically promoted apoptosis of CD138+ but not CD138- T cells to suppress autoimmune T cell accumulation in MRL/lpr mice. CONCLUSIONS: Celastrol exerted therapeutic effects on lupus by specifically promoting apoptosis of autoimmune T cells and preventing the progression of autoimmune response.

CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice.

Autoreactive T cells, specifically CD138+ (syndecan-1) T cells produced in Fas-deficient systemic lupus erythematosus (SLE) mouse models, were shown to significantly promote the generation of autoantibodies. In the present study, Murphy Roths Large lymphoproliferative (MRL/lpr) lupus mice were used to investigate the role of CD138 protein expression in T cells in the progression of SLE. Measurement of flow cytometry, immunofluorescence and Luminex were performed to determine the effect of CD138 on T cells in MRL/lpr mice. The results demonstrate that CD138+ T cells induce apoptosis via a Fas-dependent pathway. CD138 protein expression in T cells of MRL/lpr mice significantly reduced T cell apoptosis and contributed to the accumulation of T cells and double negative (DN) T cells, whilst simultaneously promoting T cell activation in Fas-deficient lupus mice. CD138 protein expression in DN T cells also significantly increased the protein expression of Fas ligand to enhance the cytotoxicity of DN T cells. Furthermore, phorbol 12-myristate 13-acetate and ionomycin (PI) stimulation reduced CD138 protein expression in CD3+ T cells and prevented CD138+ T cell accumulation by inducing specific apoptosis. PI stimulation also activated T cells in MRL/lpr mice to increase CD69 protein expression. CD69 protein expression in CD138+ T cells significantly increased the frequency of apoptotic CD138+ T cells. In addition, results from the present study demonstrated that CD138- T cells of MRL/lpr lupus mice had an activation defect. CD138 protein expression in T cells significantly reversed the defective activation and activating T cells could significantly reduce CD138 protein expression in CD3+ T cells of MRL/lpr mice. This suggests that CD138 protein expression in CD3+CD138- T cells of MRL/lpr mice may be a consequence of the impaired activation in autoreactive T cells prior to exposure to self-antigens by the immune system. CD138 expression in autoreactive T cells has a central role in promoting the progression and development of autoimmune response in MRL/lpr mice.

Glucocorticoid prevents CD138 expression in T cells of autoimmune MRL/lpr mice.

CD138+ T cells, the majority of which are CD4 and CD8 double­negative (DN) T cells, contribute to the production of anti­dsDNA antibodies in a CD4 receptor­dependent way to promote the development of systemic lupus erythematosus (SLE). Accumulation of CD138+ T cells in the spleen of MRL/lpr mice was significantly reduced by prednisone. Reduced expression of CD138 in DN T cells induced by prednisone treatment alleviated the accumulation of DN T cells in MRL/lpr mice. The frequency of CD138+ cells in CD4+ T cells of prednisone­treated MRL/lpr mice was also significantly reduced, which subsequently contributed to reduced production of anti­dsDNA antibody in the prednisone­treated MRL/lpr mice. Additionally, prednisone significantly reduced serum IgG and IgG subsets and simultaneously increased IgM secretion in serum. This suggested that glucocorticoids played a protective role during SLE treatment in MRL/lpr mice by promoting the production of IgM. The present study provides new insights into the mechanism of glucocorticoid for the treatment of SLE.

Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells.

Coxsackievirus A10 (CV-A10) is a major pathogen that causes hand, foot, and mouth disease. There are no effective therapeutic drugs for CV-A10 infection; therefore, CV-A10 vaccines should be developed. Previously, we isolated a CV-A10 strain (N25) that can be cultured on Vero cells. In this study, the N25 strain was plaque-purified three times from Vero cells, and three clones were selected for adaptive culture. The three clones of the 5th, 12th, and 19th generations were compared and analyzed in terms of viral titers, plaque morphology, pathogenicity in suckling mice, and nucleotide and amino acid sequences of the complete genome. The infectivity titers of the three clones (P2-P22) were maintained at 6.5-7.0 lgCCID50 /ml. The three clones began to proliferate at 6 h and peaked at 36 h; the corresponding CCID50 was in the range of 106.5 -106.875 /ml, which gradually decreased. The suckling mice in the challenged group exhibited clinical symptoms such as paralysis of the limbs, which gradually worsened until death. The inactivated vaccines prepared using the three clones efficiently induced antigen-specific serum antibodies in mice. There were eight nucleotide mutations in the three clones, which resulted in two and four amino acid substitutions in the VP3 and VP1 coding regions, respectively. The nucleotide and amino acid sequence homology between the three clones and N25 were 99.92%-100% and 99.78%-100%, respectively, indicating high genetic stability. Our findings provide a theoretical basis for screening CV-A10 vaccine candidate clones.

Three doses of prototypic SARS-CoV-2 inactivated vaccine induce cross-protection against its variants of concern.

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19.

LangChuangHeJi decoction ameliorates lupus via preventing accumulation of CD138+ T cells in MRL/lpr mice.

LangChuangHeJi (LCHJ) decoction has been used as a supplementary therapy to reduce the dose of prednisone and improve the therapeutic effects in systemic lupus erythematosus (SLE) maintenance. We aimed to explore the underlying mechanisms of the therapeutic effects of LCHJ. Spleen and lymph node weight, renal tissue histology, anti-dsDNA and anti-nuclear antibody levels in serum, and urinary protein levels were measured to evaluate the therapeutic effects. We further measured serum levels of multiple cytokines and antibody subsets, and performed flow cytometry analysis to observe effects of LCHJ on the frequency and activation of T cells and T cell subsets, as well as accumulation of plasma cells in splenocytes of MRL/lpr mice. LCHJ exhibited significant therapeutic effects on MRL/lpr mice. LCHJ significantly controlled the in vivo inflammation and dramatically prevented the accumulation of DN T and plasma cells in MRL/lpr mice. Moreover, LCHJ significantly suppressed the accumulation of CD138+ T cells in MRL/lpr mice, which led to the decreased production of the anti-dsDNA antibody in vivo. LCHJ significantly decreased CD4+CD138+ T cells originated from CD4+CD138- T cells, which subsequently prevented the accumulation of CD138+ T cells in MRL/lpr mice. Our results indicated that LCHJ alleviated renal injuries and prevented the enlargement of the spleen and lymph nodes by suppressing DN T cell accumulation, and reduced anti-dsDNA antibody secretion by preventing the accumulation of CD138+ T cells.

Proliferation characteristics of coxsackievirus A10 in mice and immune protection ability of experimental inactivated vaccine.

Coxsackievirus A10 (CVA10) is the main pathogen of hand, foot, and mouth disease in China. However, there are no CVA10-specific drugs and vaccines, and the pathogenesis and effects of this virus in the body are unknown. We investigated the effect of a clinically isolated CVA10 virus strain (CVA10-25) to investigate its effect in suckling mice through different infection routes. We observed the dynamic distribution and proliferation of the virus in mouse tissues by infecting suckling mice with different doses of the virus and mice of different ages with the same dose of the virus. We also analysed the pathological characteristics after infection. A formaldehyde-inactivated experimental vaccine was prepared to immunise 5-week-old BALB/c female mice three times, and newborn suckling mice were tested for the presence of maternally transmitted antibodies. The viral load in each organ after intracerebral administration was higher than that after intraperitoneal administration; the peroral administration route did not cause disease in mice. Mouse paralysis and death after infection were related to age. The skeletal muscles, heart, and lung showed histopathological changes after infection. We established a 2-day-old BALB/c suckling mouse model that could be infected intracranially to study the pathogenesis and pathology of CVA10. Maternally transmitted antibodies protected the mice against the virus. This study provides a reference for CVA10-related pathogenesis and vaccine research.

Preclinical safety assessment of a combined vaccine against Hepatitis a virus and enterovirus 71.

Since 2007, Hepatitis A (HAV) vaccination has been a part of the National Immunization Program of China. Recognizing enterovirus 71 (EV71) as the most important pathogen in severe hand, foot and mouth disease, an inactivated EV71 vaccine was successfully marketed in 2015. Based on the concept of one vaccine preventing two diseases and owing to similarities in vaccine preparation and the overlap of the eligible population, a combination of the inactivated HAV vaccine and inactivated EV71 vaccine is theoretically feasible and desirable. However, the optimal vaccinationschedule for this combination vaccine has yet to be optimized. Use of this combined vaccine would not only decrease the number of vaccinations, but also lower associated cost. This study aimed to investigate the toxicity and adverse reactions of the combined HAV-EV71 vaccine under Good Laboratory Practice conditions to provide a reference for clinical studies/applications in the future. CD®(Sprague Dawley) IGS rats were employed for single-dose toxicity testing using a high dose, and repeated-dose toxicity testing using high, as well as low doses. Animals that received only a single dose showed no obvious clinical symptoms nor abnormal body weight, and no significant gross pathological change at the experimental endpoint at necropsy. In the rats injected with three doses, phagocytosis of basophilic granules by macrophages was observed in the inguinal, mesenteric, and local lymph nodes, besides irritation at the administration site. At 56 days after the last dose, no significant histopathological change was observed in the lymph nodes, and local irritation gradually faded. Further, systematic allergy testing was performed in guinea pigs. After systemic sensitization and challenge with the HAV-EV71 vaccine, animals showed normal weight gain and no allergic reactions. This study, therefore, confirmed a good safety profile of the inactivated HAV and EV71 combined vaccine.

A comparative study on biological characteristics of ten coxsackievirus A10 virus strains.

In this study, we analyzed ten CVA10 strains were genotyped and cultured for 10 generations to detect plaque morphology, pathogenicity, growth and other characteristics. Mice were injected with live and inactivated virus to detect neutralizing antibody titers. The results suggested that all CVA10 strains fell into Genotype C. Each strain cultured on KMB17 and Vero cells, increased from 1st generation onwards to peak in the 3rd and 4th, and the titer at which each became infectious ranged from 5.0 to 6.5 and 6.0 to 7.0 lgCCID50/ml, respectively. Two-day-old BALB/c mice were selected and inoculated intracerebral with the CVA10 strains, Limb paralysis was significant as early as 3 d; paralysis of all limbs for 50% of affected mice. LT50 was approximately 6 d, all died within 8 d. Ten strains induced good immune response, the GMT value of booster immunizations was higher than that of initial immunization. This provide reference points for selecting CVA10 vaccine candidates.

N-terminally truncated nucleocapsid protein of SARS-CoV-2 as a better serological marker than whole nucleocapsid protein in evaluating the immunogenicity of inactivated SARS-CoV-2.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.

Immune responses of a CV-A16 live attenuated candidate strain and its protective effects in rhesus monkeys.

Coxsackievirus A16 (CV-A16) is a major causative pathogen of hand, foot, and mouth diseases (HFMDs). The licensed HFMD vaccine targets EV-A71 without cross-protection against CV-A16. Thus, a CV-A16 vaccine is needed. In this study, the immunogenicity and protective efficacy of a live attenuated CV-A16 candidate, K168-8Ac, were evaluated in a rhesus monkey model. Four passages of this strain (P35, P50, P60, and P70) were administered to monkeys, and its protective effect was identified. The immunized monkeys were clinically asymptomatic, except for slight fever. Weak viraemia was observed, and two doses of vaccination were found to significantly reduce virus shedding. High levels of antibody responses were observed (1:1024-1:2048), along with a significant increase in plasma IL-8. The I.M. group showed a much stronger humoural immunity. Pathological damage was detected mainly in lung tissues, although thalamus, spinal cord, lymph nodes, and livers were involved. After the viral challenge, it was found that two doses of vaccine reduced virus shedding, and the degree of lung damage and the number of organs involved decreased as the passage number increased. Overall, a robust immune response and partial protection against CV-A16, triggered by the K168-8Ac strain, were demonstrated. This study provides valuable data for CV-A16 vaccine development.

Safety and immunogenicity of an experimental live combination vaccine against enterovirus 71 and coxsackievirus A16 in rhesus monkeys.

Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the two most common pathogens causing hand, foot, and mouth disease (HFMD). Previously, we obtained one candidate live attenuated strain each for EV-A71 and CV-A16; here, we evaluated the safety and immunogenicity of a combinedlive vaccine against EV-A71 and CV-A16 generated from these two candidate strains. Rhesus monkeys were intramuscularly treated with a live combinationvaccine against both EV-A71 and CV-A16 or with either vaccine alone. No fever or atypical clinical signs were observed in any animals. Monkeys vaccinated with the combinationlive vaccine presented no notable pathological changes in the brain, spinal cord, lung, and liver; in contrast, these regions showed inflammatory cell infiltration in monkeys treated with EV-A71 alone or CV-A16 alone. Weak viremia was detected in plasma after inoculation with the combinationvaccine; however, the duration of viral shedding in feces was increased. Biochemical studies revealed a slight increase in aspartate aminotransferase levels in monkeys inoculated with the live combination vaccine; however, histopathological findings did not attribute this change to liver damage. We also found that the live combinationvaccine induced a dual humoral immune response. Cytokine analysis indicated that the combined EV-A71/CV-A16 vaccine significantly down-regulated interleukin-8 production. Here, we have demonstrated that the live attenuated EV-A71/CV-A16 vaccine was safe and could trigger a dual specific immune response. However, its immune protection efficacy requires further investigation.

PTP1B negatively regulates nitric oxide-mediated Pseudomonas aeruginosa killing by neutrophils.

Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-ß, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction.

The inhibitory effects of Qingchang Wenzhong granule on the interactive network of inflammation, oxidative stress, and apoptosis in rats with dextran sulfate sodium-induced colitis.

BACKGROUND: Ulcerative colitis (UC) is a kind of complex immune disease, the pathogenesis of which remains elusive. Destruction of the intestinal barrier, extreme inflammation, oxidative stress, and apoptosis might play key roles in the development of UC. In previous studies, we observed that Qingchang Wenzhong granule (QCWZG) had the exact effect on the remission of UC in the clinic; however, the underlying mechanism has not been identified. This study aimed to reveal the effects of QCWZG on the intestinal physical barrier and the interactive network of inflammation, oxidative stress, and apoptosis in rats with dextran sulfate sodium (DSS)-induced colitis. METHODS: Sixty rats were randomly divided into six groups: blank group, model group, high/mild/low-dose QCWZG groups, and mesalazine group. The rats in the experimental group drank 4% DSS for 7 days and 1% DSS for the subsequent 7 days. Different medications or distilled water was supplied by intragastric administration for 7 days. The levels of colitis and indices related to inflammation, oxidative stress, and apoptosis were assessed. RESULTS: Compared with the model group, the QCWZG group (P < 0.05) demonstrated attenuated disease activity index, colonic mucosa disease index, histological lesions, and colonic weights; lower levels of inflammatory substances, such as interleukin (IL)-1α, IL-6, tumor necrosis factor-α, and myeloperoxidase; lower levels of malondialdehyde; and increased levels of superoxide dismutase and glutathione peroxidase. The QCWZG group also demonstrated elevated expression of Bcl-2 and occluding but downregulated db expression of Bax and caspase 3 in the colon. CONCLUSION: QCWZG could relieve rats with DSS-induced colitis from UC symptoms by improving the intestinal physical barrier, which resists the interactive network of inflammation, oxidative stress, apoptosis, and their overactivated interactions.

CXCL4 contributes to host defense against acute Pseudomonas aeruginosa lung infection.

Platelets have been implicated in pulmonary inflammation following exposure to bacterial stimuli. The mechanisms involved in the platelet-mediated host response to respiratory bacterial infection remain incompletely understood. In this study, we demonstrate that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) plays critical roles in a mouse model of acute bacterial pneumonia using Pseudomonas aeruginosa. Platelets are activated during P. aeruginosa infection, and mice depleted of platelets display markedly increased mortality and impaired bacterial clearance. CXCL4 deficiency impairs bacterial clearance and lung epithelial permeability, which correlate with decreased neutrophil recruitment to BALF. Interestingly, CXCL4 deficiency selectively regulates chemokine production, suggesting that CXCL4 has an impact on other chemokine expression. In addition, CXCL4 deficiency reduces platelet-neutrophil interactions in blood following P. aeruginosa infection. Further studies revealed that platelet-derived CXCL4 contributes to the P. aeruginosa-killing of neutrophils. Altogether, these findings demonstrate that CXCL4 is a vital chemokine that plays critical roles in bacterial clearance during P. aeruginosa infection through recruiting neutrophils to the lungs and intracellular bacterial killing.

Qingchang Wenzhong Decoction Attenuates DSS-Induced Colitis in Rats by Reducing Inflammation and Improving Intestinal Barrier Function via Upregulating the MSP/RON Signalling Pathway.

Ulcerative colitis (UC) is a chronic, nonspecific, inflammatory disease for which an effective treatment is lacking. Our previous study found that Qingchang Wenzhong Decoction (QCWZD) can significantly improve the clinical symptoms of UC and ameliorate dextran sulphate sodium- (DSS-) induced ulcerative colitis in rats by downregulating the IP10/CXCR3 axis-mediated inflammatory response. The purpose of the present study was to further explore the mechanism of QCWZD for UC in rats models, which were established by 7-day administration of 4.5% dextran sulphate sodium solution. QCWZD was administered daily for 7 days; then we determined the serum macrophage-stimulating protein concentration (MSP) and recepteur d'origine nantais (RON) expression and its downstream proteins (protein kinase B [Akt], phosphorylated [p] Akt, occludin, zona occluden- [ZO-] 1, and claudin-2) in colon tissue using Western blotting and quantitative polymerase chain reaction. In DSS-induced UC, QCWZD significantly alleviated colitis-associated inflammation, upregulated serum MSP expression and RON expression in the colon, reduced the pAkt levels, promoted colonic occluding and ZO-1 expression, and depressed claudin-2 expression. In conclusion, the MSP/RON signalling pathway plays an important role in the pathogenesis of UC by involving the inflammatory response and improving intestinal barrier function. QCWZD appears to attenuate DSS-induced UC in rats by upregulating the MSP/RON signalling pathway.

A comparative study of multiple clinical enterovirus 71 isolates and evaluation of cross protection of inactivated vaccine strain FY-23 K-B in vitro.

BACKGROUND: Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease, which mostly affects infants and children and leads to severe neurological diseases. Vaccination offers the best option for disease control. We have screened the virus strain FY-23 K-B, which is used as an inactivated vaccine strain. An important issue in the development of vaccines is whether they provide cross protection against all other strains. METHODS: We collected and identified 19 clinical EV71 isolates from mainland China, which all belong to the C4 genotype. We established growth curves of the strains in Vero cells, performed genetic analysis, and evaluated the cross protection efficacy through neutralizing assays using antisera from a rabbit, monkey and adult human immunized with the FY-23 K-B vaccine strain. RESULTS: The antisera showed broad cross protection among the C4 subgroup strains and homotype strain. Neutralizing indexes (NIs) among the isolates and homotype strain of antisera varied between 56.2-1995.3 for rabbit, 17.8-42,169.7 for monkey and 31.6-17,782.8 for human, whereas NIs against Coxsackievirus A16 or other enteroviruses were below 10. CONCLUSIONS: These results suggested that FY-23 K-B used as an antigen could elicit broad spectrum neutralizing antibodies with cross protective efficacy among C4 genotype strains.

Yinchen Linggui Zhugan Decoction Ameliorates Nonalcoholic Fatty Liver Disease in Rats by Regulating the Nrf2/ARE Signaling Pathway.

Yinchen Linggui Zhugan Decoction (YCLGZGD) is the combination of Linggui Zhugan (LGZGD) and Yinchenhao (YCHD) decoctions, two famous traditional Chinese medicine prescriptions. In previous studies, we found that Yinchen Linggui Zhugan Decoction (YCLGZGD) could regulate lipid metabolism disorder and attenuate inflammation in pathological process of nonalcoholic fatty liver disease (NAFLD). However, the exact underlying mechanism remains unknown. The aim of this study was to explore the effect of Yinchen Linggui Zhugan Decoction on experimental NAFLD and its mechanism in rats with high-fat diet (HFD) which was established by 8-week administration of HFD. YCLGZGD, LGZGD, and YCHD were administered daily for 4 weeks, after which the rats were euthanized. The level of blood lipid, liver enzymes, H&E, and Oil Red O staining were determined to evaluate NAFLD severity. Western blotting and real-time polymerase chain reaction were, respectively, used to determine hepatic protein and gene expression of Keap1, Nrf2, NQO1, and HO-1. Oral YCLGZGD ameliorated HFD-induced NAFLD. Furthermore, YCLGZGD increased the protein and gene expression of Nrf2, NQO1, and HO-1 without changing Keap1. Overall, these results suggest that YCLGZGD ameliorates HFD-induced NAFLD in rats by upregulating the Nrf2/ARE signaling pathway.