SMEK1 ablation promotes glucose uptake and improves obesity-related metabolic dysfunction via AMPK signaling pathway.

Obesity has become a major risk of global public health. SMEK1 is also known as a regulatory subunit of protein phosphatase 4 (PP4). Both PP4 and SMEK1 have been clarified in many metabolic functions, including the regulation of hepatic gluconeogenesis and glucose transporter gene expression in yeast. Whether SMEK1 participates in obesity and the broader metabolic role in mammals is unknown. Thus, we investigated the function of SMEK1 in white adipose tissue and glucose uptake. GWAS/GEPIA/GEO database was used to analyze the correlation between SMEK1 and metabolic phenotypes/lipid metabolism-related genes/obesity. Smek1 KO mice were generated to identify the role of SMEK1 in obesity and glucose homeostasis. Cell culture and differentiation of stromal-vascular fractions (SVFs) and 3T3-L1 were used to determine the mechanism. 2-NBDG was used to measure the glucose uptake. Compound C was used to confirm the role of AMPK. We elucidated that SMEK1 was correlated with obesity and adipogenesis. Smek1 deletion enhanced adipogenesis in both SVFs and 3T3-L1. Smek1 KO protected mice from obesity and had protective effects on metabolic disorders, including insulin resistance and inflammation. Smek1 KO mice had lower levels of fasting serum glucose. We found that SMEK1 ablation promoted glucose uptake by increasing p-AMPKα(T172) and the transcription of Glut4 when the effect on AMPK-regulated glucose uptake was due to the PP4 catalytic subunits (PPP4C). Our findings reveal a novel role of SMEK1 in obesity and glucose homeostasis, providing a potential new therapeutic target for obesity and metabolic dysfunction.NEW & NOTEWORTHY Our study clarified the relationship between SMEK1 and obesity for the first time and validated the conclusion in multiple ways by combining available data from public databases, human samples, and animal models. In addition, we clarified the role of SMEK1 in glucose uptake, providing an in-depth interpretation for the study of its function in glucose metabolism.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Microglial Pdcd4 deficiency mitigates neuroinflammation-associated depression via facilitating Daxx mediated PPARγ/IL-10 signaling.

The dysregulation of pro- and anti-inflammatory processes in the brain has been linked to the pathogenesis of major depressive disorder (MDD), although the precise mechanisms remain unclear. In this study, we discovered that microglial conditional knockout of Pdcd4 conferred protection against LPS-induced hyperactivation of microglia and depressive-like behavior in mice. Mechanically, microglial Pdcd4 plays a role in promoting neuroinflammatory responses triggered by LPS by inhibiting Daxx-mediated PPARγ nucleus translocation, leading to the suppression of anti-inflammatory cytokine IL-10 expression. Finally, the antidepressant effect of microglial Pdcd4 knockout under LPS-challenged conditions was abolished by intracerebroventricular injection of the IL-10 neutralizing antibody IL-10Rα. Our study elucidates the distinct involvement of microglial Pdcd4 in neuroinflammation, suggesting its potential as a therapeutic target for neuroinflammation-related depression.

Smek1 deficiency exacerbates experimental autoimmune encephalomyelitis by activating proinflammatory microglia and suppressing the IDO1-AhR pathway.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal disease model of multiple sclerosis (MS) that involves the immune system and central nervous system (CNS). However, it is unclear how genetic predispositions promote neuroinflammation in MS and EAE. Here, we investigated how partial loss-of-function of suppressor of MEK1 (SMEK1), a regulatory subunit of protein phosphatase 4, facilitates the onset of MS and EAE. METHODS: C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to establish the EAE model. Clinical signs were recorded and pathogenesis was investigated after immunization. CNS tissues were analyzed by immunostaining, quantitative polymerase chain reaction (qPCR), western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Single-cell analysis was carried out in the cortices and hippocampus. Splenic and lymph node cells were evaluated with flow cytometry, qPCR, and western blot analysis. RESULTS: Here, we showed that partial Smek1 deficiency caused more severe symptoms in the EAE model than in controls by activating myeloid cells and that Smek1 was required for maintaining immunosuppressive function by modulating the indoleamine 2,3-dioxygenase (IDO1)-aryl hydrocarbon receptor (AhR) pathway. Single-cell sequencing and an in vitro study showed that Smek1-deficient microglia and macrophages were preactivated at steady state. After MOG35-55 immunization, microglia and macrophages underwent hyperactivation and produced increased IL-1ß in Smek1-/+ mice at the peak stage. Moreover, dysfunction of the IDO1-AhR pathway resulted from the reduction of interferon γ (IFN-γ), enhanced antigen presentation ability, and inhibition of anti-inflammatory processes in Smek1-/+ EAE mice. CONCLUSIONS: The present study suggests a protective role of Smek1 in autoimmune demyelination pathogenesis via immune suppression and inflammation regulation in both the immune system and the central nervous system. Our findings provide an instructive basis for the roles of Smek1 in EAE and broaden the understanding of the genetic factors involved in the pathogenesis of autoimmune demyelination.

Lack of WDFY4 Aggravates Ovalbumin-Induced Asthma via Enhanced Th2 Cell Differentiation.

BACKGROUND: Asthma is a chronic inflammatory airway disease, and Th2 cells play an important role in asthma. WDFY4 (WDFY family member 4) is a susceptibility gene in several autoimmune diseases. OBJECTIVE: In this study, the roles of WDFY4 in Th2 cell differentiation and Th2-dependent asthma were investigated. METHODS: Naïve CD4+ T cells were isolated from wild-type and WDFY4-deficient mice and induced to differentiate in vitro. Subsequently, a mouse model of asthma was established by sensitization with ovalbumin. RESULTS: Study results showed that WDFY4 deficiency could promote the differentiation of Th2 cells and the production of Th2 cytokines. WDFY4-deficient asthmatic mice showed higher levels of Th2 cytokines in the lungs and bronchoalveolar lavage fluid than wild-type mice. Moreover, infiltration of inflammatory cells, hyperplasia of goblet cells, production of mucus, and deposition of collagen were enhanced in WDFY4-deficient asthmatic mice. CONCLUSIONS: Our study demonstrates the pivotal role of WDFY4 in the pathogenesis of asthma and in Th2 cell differentiation.

Correction to: A De Novo Frameshift Mutation in TNFAIP3 Impairs A20 Deubiquitination Function to Cause Neuropsychiatric Systemic Lupus Erythematosus.

Figure 4a in Manuscript ID#JOCI-D-19-00318 has been revised due to the replacement of immunoblot lane of ß-catenin by Zo-1 in NHA group.

WDFY4 Is Involved in Symptoms of Systemic Lupus Erythematosus by Modulating B Cell Fate via Noncanonical Autophagy.

Genome-wide association studies have recently illuminated that WDFY4 is genetically associated with systemic lupus erythematosus (SLE) susceptibility in various ethnic groups. Despite strong genetic evidence suggesting a role of WDFY4 in SLE pathogenesis, its functional relevance is largely unknown. In this study, we generated Wdfy4 B lymphocyte conditional knockout (Wdfy4-CKO) mice and found that loss of Wdfy4 led to a decrease in number of total B cells and several subpopulations of B cells in the periphery and a defect in the transition from the pro- to pre-B cell stage in bone marrow. Also, Wdfy4-CKO mice showed impaired Ab responses as compared with controls when challenged with Ag. SLE phenotypes were effectively alleviated in Wdfy4-CKO mice, with significantly diminished pristane-elicited production of autoantibodies and glomerulonephritis. Genetic silencing of WDFY4 in B cells increased lipidation of LC3 independent of p62 and Beclin1, which are essential proteins of canonical autophagy. Our in vivo and in vitro data suggest that WDFY4 facilitates noncanonical autophagic activity. Our findings provide a novel functional link underlying the mechanism of SLE in which WDFY4 influences B cell fate via noncanonical autophagy.

[Analysis of deafness gene variant screening of 7875 neonatal cases in Dongying area of Shandong].

OBJECTIVE: To determine the types and frequency of deafness-related variants among 7875 newborns from Dongying area of Shandong Province. METHODS: One hundred loci of 18 common deafness genes were subjected to semiconductor sequencing. Variant site, frequency and distribution of the variants were analyzed. RESULTS: In total 552 deafness gene variants were detected among the 7875 newborns, which yielded a detection rate of 7.01%. Among these, common variant sites for GJB2, SLC26A4 and GJB3 genes were c.235delC, IVS7-2A>G and c.538C>T, respectively. The variant frequencies of matrilinear inheritance deafness genes MT-CO1, MT-RNR1, MT-TL1 and MT-TS1 were 0.38%, 0.25%, 0.1% and 0.01%, respectively. Four newborns were diagnosed with deafness, among which one had unilateral hearing loss. Analysis of the proportions of neonatal deafness-related variants in five counties of Dongying showed that the highest variant rate for the SLC26A4 gene compared with GJB2 was in Lijin county (51.76% vs. 40%), while the lowest was in Hekou county (30.77% vs. 56.41%). CONCLUSION: The carrier rate of deafness-related variants in Dongying area is higher than other regions of China, which may be attributed to the increased types and variant sites covered by the semiconductor sequencing method compared with the chip method and time-of-flight mass spectrometry. Due to geographical and population aggregation factors, the proportion of deafness variants in the five counties of Dongying differed significantly. Above results may provide a guide for the prevention of congenital deafness in Dongying area.

Cervical Spinal Involvement in a Chinese Pedigree With Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy Caused by a 3' Untranslated Region Mutation of COL4A1 Gene.

Background and Purpose- Pontine autosomal dominant microangiopathy and leukoencephalopathy, a recently defined subtype of cerebral small vessel disease, is associated with mutations in COL4A1 (collagen type IV alpha 1 chain) 3' untranslated region. We here describe a pontine autosomal dominant microangiopathy and leukoencephalopathy pedigree with COL4A1 mutation presenting both pontine and cervical spinal cord involvement. Methods- For the diagnostic purpose, brain and spinal magnetic resonance imaging scanning, skin biopsy, and whole-exome sequencing were performed on the patients in the pedigree. Suspected pathogenic variants were further confirmed by cosegregation analysis using Sanger sequencing in the family members. Results- We identified a mutation located at the binding site of miR-29 (microRNA-29) in 3' untranslated region of COL4A1(c.*32G>A). The pontine autosomal dominant microangiopathy and leukoencephalopathy patients in this pedigree carried this variant, whereas other healthy family members but one did not. Magnetic resonance imaging showed lesions in the pons, white matter, and cervical spinal cord. Skin biopsy revealed thickened basal lamina in vessels. Conclusions- For the first time, we reported cervical spinal involvement in pontine autosomal dominant microangiopathy and leukoencephalopathy and expanded the clinical spectrum of this disease.

A De Novo Frameshift Mutation in TNFAIP3 Impairs A20 Deubiquitination Function to Cause Neuropsychiatric Systemic Lupus Erythematosus.

PURPOSE: Genome-wide association study of systemic lupus erythematosus (SLE) revealed tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) as a susceptibility gene. Here, we report a de novo mutation in TNFAIP3 in a Chinese patient with neuropsychiatric SLE (NPSLE). METHODS: Whole exome sequencing was performed for the patient and healthy members from the family. Suspected pathogenic variants were further analyzed and co-segregation was confirmed by Sanger sequencing. Real-time PCR and western blot were performed with peripheral blood mononuclear cells (PBMCs) and patient-derived T cells. Transfected HEK293T cells, human umbilical vein endothelial cells, normal human astrocytes, and microglia were used for in vitro studies. RESULTS: A de novo frameshift mutation in TNFAIP3 was found in the NPSLE patient. Western blot analysis showed activated NF-κB and mitogen-activated protein kinase pathways. Real-time PCR revealed elevated expression of pro-inflammatory cytokines. On immunoprecipitation assay, the mutant A20 altered the K63-linked ubiquitin level of TRAF6 via its ubiquitin-editing function. CONCLUSIONS: The mutant A20 may play a role in weakening the tight junction of the blood-brain barrier to cause neurologic symptoms. We report a rare variant of TNFAIP3 in a patient with NPSLE and reveal its autoimmune disease-causing mechanism in both peripheral tissues and the central nervous system.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

ORMDL3 Facilitates the Survival of Splenic B Cells via an ATF6α-Endoplasmic Reticulum Stress-Beclin1 Autophagy Regulatory Pathway.

The genetic association of orosomucoid-like 3 (ORMDL3) with an array of immunoinflammatory disorders has been recently unraveled in multiple ethnic groups, and functional exploration has received attention of the particular relevance of this gene in endoplasmic reticulum stress, lipid metabolism, and inflammatory response. In this study, we demonstrated the upregulation of ORMDL3 in both patients with systemic lupus erythematosus and lupus mice compared with controls. By establishing ORMDL3 knockout mice (Ormdl3-/-), we showed that silencing Ormdl3 in vivo significantly decreased the proportions of mature B lymphocytes and transitional 2B cells in spleen and B1a cells from abdominal cavity perfusion fluid, the secretion of IgG and IgM, and the expression of Baff. Additionally, knockdown of Ormdl3 augmented the apoptosis of total splenic cells and splenic CD19+ B cells but did not affect B cell proliferation and cell cycle. Subsequently, we in vitro and in vivo demonstrated that ORMDL3 potentially mediates the autophagy via the ATF 6-Beclin1 autophagy pathway, and it facilitates the survival of splenic B cells via promoting autophagy and suppressing apoptosis. Taken together, we uncovered a role of ORMDL3 in fine-tuning B cell development and survival, besides highlighting a potential mechanism by which ORMDL3 regulates autophagy via ATF6 pathway.

Ankylosing spondylitis: analysis of gene-gene interactions between IL-12ß, JAK2, and STAT3 in Han Chinese and Algerian cohorts.

INTRODUCTION: Association studies have recently identified the importance of new genetic variants for ankylosing spondylitis (AS) in several populations. Our aim was to confirm associations of variants within genes involved in the IL-23 signalling pathway with AS in two ethnically different populations: Han Chinese and Algerian. MATERIAL AND METHODS: Two case-control studies were performed in separate cohorts: Han Chinese (430 AS patients and 580 controls) and Algerian (130 AS patients and 120 controls). We genotyped four single nucleotide polymorphisms (SNPs): rs3212227 (or +1188A/C) and rs6887695 in IL-12ß, rs7857730 in JAK2, and rs2293152 in STAT3, using TaqMan SNP genotyping assays. Gene-gene interaction analyses were also tested by logistic regression and multifactor dimensionality reduction (MDR). RESULTS: Statistical analysis revealed a difference in allele frequencies between AS patients and controls for rs321222 in the IL-12ß gene in both the Han Chinese (p = 0.005) and the Algerian (p = 0.031) cohorts. Two other associations were reported with JAK2 rs7857730 in the Han Chinese (allelic p = 0.014) cohort and STAT3 rs2293152 in the Algerian (allelic p = 0.006) cohort. Moreover, logistic regression analyses showed a number of significant combinations within the two populations, and the gene-gene epistasis effects in AS were also confirmed by MDR. CONCLUSIONS: Our findings have confirmed the association between genes in IL-23 signalling pathway and the pathogenesis of AS. This association was particularly novel in both Han Chinese and Algerian populations with the 3' untranslated region (3'UTR) variant rs3212227 (or +1188A/C) of IL-12ß. The gene-gene interaction models in this pathway may thus increase the risk of AS in these populations.

Whole-Genome Linkage Analysis with Whole-Exome Sequencing Identifies a Novel Frameshift Variant in NEFH in a Chinese Family with Charcot-Marie-Tooth 2: A Novel Variant in NEFH for Charcot-Marie-Tooth 2.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is the most common neurodegenerative disorder of the peripheral nervous system. More than 50 genes/loci were found associated with the disease. We found a family with autosomal-dominant CMT2. OBJECTIVE: To reveal the pathogenic gene of the family and further investigate the function of the variant. METHODS: DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members. Neurofilament light polypeptide and wild-type or mutant neurofilament heavy polypeptide (NEFH) were co-transfected into SW13 (vim-) cells. The nefh-knockdown zebrafish model was produced by using morpholino antisense oligonucleotides. RESULTS: We identified a novel insertion variant (c.3057insG) in NEFH in the family. The variant led to the loss of a stop codon and an extended 41 amino acids in the protein. Immunofluorescence results revealed that mutant NEFH disrupted the neurofilament network and induced aggregation of NEFH protein. Knockdown of nefh in zebrafish caused a slightly or severely curled tail. The motor ability of nefh-knockdown embryos was impaired or even absent, and the embryos showed developmental defects of axons in motor neurons. The abnormal phenotype and axonal developmental defects could be rescued by injection of human wild-type but not human mutant NEFH mRNA. CONCLUSIONS: We identified a novel stop loss variant in NEFH that is likely pathogenic for CMT2, and the results provide further evidence for the role of an aberrant assembly of neurofilament in CMT.

Zebrafish cul4a, but not cul4b, modulates cardiac and forelimb development by upregulating tbx5a expression.

CUL4A and CUL4B are closely related cullin family members and can each assemble a Cullin-RING E3 ligase complex (CRL) and participate in a variety of biological processes. While the CRLs formed by the two cullin members may have common targets, the two appeared to have very different consequences when mutated or disrupted in mammals. We here investigated the roles of cul4a and cul4b during zebrafish embryogenesis by using the morpholino knockdown approach. We found that cul4a is essential for cardiac development as well as for pectoral fin development. Whereas cul4a morphants appeared to be unperturbed in chamber specification, they failed to undergo heart looping. The failures in heart looping and pectoral fin formation in cul4a morphants were accompanied by greatly reduced proliferation of cardiac cells and pectoral fin-forming cells. We demonstrated that tbx5a, a transcription factor essential for heart and limb development, is transcriptionally upregulated by cul4a and mediates the function of cul4a in cardiac and pectoral fin development. In contrast to the critical importance of cul4a, cul4b appeared to be dispensable for zebrafish development and was incapable of compensating for the loss of cul4a. This work provides the first demonstration of an essential role of cul4a, but not cul4b, in cardiac development and in the regulation of tbx5a in zebrafish. These findings justify exploring the functional role of CUL4A in human cardiac development.

miR-155 Deficiency Ameliorates Autoimmune Inflammation of Systemic Lupus Erythematosus by Targeting S1pr1 in Faslpr/lpr Mice.

MicroRNA-155 (miR-155) was previously found involved in the development of systemic lupus erythematosus (SLE) and other autoimmune diseases and the inflammatory response; however, the detailed mechanism of miR-155 in SLE is not fully understood. To explore the in vivo role of miR-155 in the pathogenesis of SLE, miR-155-deficient Fas(lpr/lpr) (miR-155(-/-)Fas(lpr/lpr)) mice were obtained by crossing miR-155(-/-) and Fas(lpr/lpr) mice. Clinical SLE features such as glomerulonephritis, autoantibody levels, and immune system cell populations were compared between miR-155(-/-)Fas(lpr/lpr) and Fas(lpr/lpr) mice. Microarray analysis, RT-PCR, Western blot, and luciferase reporter gene assay were used to identify the target gene of miR-155. miR-155(-/-)Fas(lpr/lpr) mice showed milder SLE clinical features than did Fas(lpr/lpr)mice. As compared with Fas(lpr/lpr) mice, miR-155(-/-)Fas(lpr/lpr) mice showed less deposition of total IgA, IgM, and IgG and less infiltration of inflammatory cells in the kidney. Moreover, the serum levels of IL-4 and IL-17a, secreted by Th2 and Th17 cells, were lower in miR-155(-/-)Fas(lpr/lpr) than Fas(lpr/lpr) mice; the CD4(+)/CD8(+) T cell ratio was restored in miR-155(-/-)Fas(lpr/lpr) mice as well. Sphingosine-1-phosphate receptor 1 (S1PR1) was found as a new target gene of miR-155 by in vitro and in vivo studies; its expression was decreased in SLE patients and Fas(lpr/lpr) mice. miR-155(-/-)Fas(lpr/lpr) mice are resistant to the development of SLE by the regulation of the target gene S1pr1. miR-155 might be a new target for therapeutic intervention in SLE.

Safety pharmacology and subchronic toxicity of jinqing granules in rats.

BACKGROUND: Jinqing granules which are made of a mixture extract that contains Radix Tinosporae and Canarii fructus in proportions according to a longstanding formula have a good effect on the prevention and treatment of gastric ulcer disease. It has not been through safety through systematic toxicological studies, however. To provide basis for clinical application, we performed safety pharmacology and subchronic toxicity experiments in specific pathogen-free Sprague-Dawley rats. RESULTS: In safety pharmacology experiments, Jinqing granules had no evident adverse effects on the central nervous, cardiovascular, or respiratory systems. In subchronic toxicity study, 2-8 g/kg of Jinqing granules induced no evident adverse effects on Clinical signs, body weight changes, food and water intake, death daily, indicators of urine, hematological assay, serum biochemistry, organ coefficient and histopathological examination. However, the 16 g/kg dose was associated with slightly slowed weight growth, decreased number of sperm in seminiferous tubules and increased values of serum aspartate aminotransferase and bilirubin. During the 30-day feeding test, 3 rats that received the 16 g/kg dose died, but the deaths were most likely due to trauma of oral gavage, not to drug toxicity. CONCLUSION: Jinqing granules given to Sprague-Dawley rats orally for 30 days at a dose of 8 g/kg or less appears safe, but higher doses were not proven safe. The significance of these observations with respect to animal usage of Jinqing granules deserves thorough investigation.