A surface-independent bioglue using photo-crosslinkable benzophenone moiety.

Surface coating technology is broadly demanded across various fields, including marine and biomedical materials; therefore, a facile and versatile approach is desired. This study proposed an attractive surface coating strategy using photo-crosslinkable benzophenone (BP) moiety for biomaterials application. BP-containing "bioglue" polymer can effectively crosslink with all kinds of surfaces and biomolecules. Upon exposure to ultraviolet (UV) light, free radical reaction from the BP glue facilitates the immobilization of diverse molecules onto different substrates in a straightforward and user-friendly manner. Through either one-step, mixing the bioglue with targeted biomolecules, or two-step methods, pre-coating the bioglue and then adding targeted biomolecules, polyacrylic acid (PAA), cyclic RGD-containing peptides, and proteins (gelatin, collagen, and fibronectin) were successfully immobilized on substrates. After drying the bioglue, targeted biomolecules can still be immobilized on the surfaces preserving their bioactivity. Cell culture on biomolecule-immobilized surfaces using NIH 3T3 fibroblasts and human bone marrow stem cells (hBMSCs) showed significant improvement of cell adhesion and activity compared to the unmodified control in serum-free media after 24 hours. Furthermore, hBMSCs on the fibronectin-immobilized surface showed an increased calcium deposition after 21 days of osteogenic differentiation, suggesting that the immobilized fibronectin is highly bioactive. Given the straightforward protocol and substrate-independent bioglue, the proposed coating strategy is promising in broad-range fields.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Engineering Cell Microenvironment Using Nanopattern-Derived Multicellular Spheroids and Photo-Crosslinked Gelatin/Hyaluronan Hydrogels.

Cell cultures of dispersed cells within hydrogels depict the interaction of the cell-extracellular matrix (ECM) in 3D, while the coculture of different cells within spheroids combines both the effects of cell-cell and cell-ECM interactions. In this study, the cell co-spheroids of human bone mesenchymal stem cells/human umbilical vein endothelial cells (HBMSC/HUVECs) are prepared with the assistance of a nanopattern, named colloidal self-assembled patterns (cSAPs), which is superior to low-adhesion surfaces. A phenol-modified gelatin/hyaluronan (Gel-Ph/HA-Ph) hydrogel is used to encapsulate the multicellular spheroids and the constructs are photo-crosslinked using blue light. The results show that Gel-Ph/HA-Ph hydrogels with a 5%-to-0.3% ratio have the best properties. Cells in HBMSC/HUVEC co-spheroids are more favorable for osteogenic differentiation (Runx2, ALP, Col1a1 and OPN) and vascular network formation (CD31+ cells) compared to HBMSC spheroids. In a subcutaneous nude mouse model, the HBMSC/HUVEC co-spheroids showed better performance than HBMSC spheroids in angiogenesis and the development of blood vessels. Overall, this study paves a new way for using nanopatterns, cell coculturing and hydrogel technology for the generation and application of multicellular spheroids.

Polydopamine-Mediated Protein Adsorption Alters the Epigenetic Status and Differentiation of Primary Human Adipose-Derived Stem Cells (hASCs).

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and ß1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Zhenzhu Xiaoji Decoction Induces Autophagy and Apoptosis Cell Death in Liver Cancer Cells through AKT/mTOR and JAK2/STAT3 Signaling Pathway.

Background: Liver cancer is one of the most common digestive tumors. The prescription Zhenzhu Xiaoji decoction (ZZXJD) has a certain effect on the growth and survival of primary liver cancer. Object: This article aimed to explore the effect and molecular mechanism of ZZXJD on liver cancer SMMC-7721 cells. Method: The research groups were divided into the model group, ZZXJD group, and cisplatin group. SMMC-7721 cells were treated with different concentrations of ZZXJD-medicated serum for 24 h and 48 h. The cell viability was measured with CCK8 assay, and cell morphology was observed by fluorescence microscope and transmission electron microscope (TEM). Western blot, RT-PCR, and gene chip were used to determine the protein expression level and gene expression level of cells and tumor tissues. Results: ZZXJD inhibited the proliferation activity of SMMC-7721 cells in a concentration- and time-dependent manner. The morphological changes of the cell showed apoptosis and autophagy. The gene expression of protein kinase B (AKT), mammalian target of rapamycin (mTOR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3(STAT3) were downregulated compared with the model group(p < 0.05). The nude mice experiments confirmed that ZZXJD inhibited the growth of tumors in tumor-bearing mice, and the effect increased with the increase of concentration. Conclusion: ZZXJD induced autophagy and apoptosis of liver cancer cells via inhibiting AKT/mTOR signaling pathway and JAK2/STAT3 signaling pathway, thereby affecting the growth and survival of liver cancer cells.

Programming Colloidal Self-Assembled Patterns (cSAPs) into Thermo-Responsible Hybrid Surfaces for Controlling Human Stem Cells and Macrophages.

Hybrid surfaces with tunable topography, chemistry, and stiffness have potential to rebuild native extracellular matrix (ECM) and manipulate cell behavior in vitro. However, the fabrication of controllable hybrid surfaces is still challenging. In this study, colloidal self-assembly technology was used to program particles into highly ordered structures with hybrid chemistry and stiffness at biointerfaces. These colloidal self-assembled patterns (cSAPs), including unary, binary, and ternary cSAPs, composed of silicon (Si), polystyrene (PS), and/or poly(N-isopropylacrylamide) (pNIPAM) nanogels (PNGs), were fabricated using either coassembly or layer-by-layer (LBL) methods. The selected binary cSAPs (i.e., PS/PNG and PNG/PS) have a tunable surface topography and wettability between 25 and 37 °C; thus, they can be used as dynamic cell culture substrates. Human adipose-derived mesenchymal stem cells (hASCs), bone marrow-derived mesenchymal stem cells (hBMSCs), and macrophages (THP-1) were investigated on these hybrid cSAPs under a static or dynamic system. The results showed that hybrid cSAPs significantly influenced the focal adhesions, cell morphology, cell migration, and gene expressions of stem cells. In general, stem cells had more vinculin puncta, smaller spreading size, and faster migration speed than the TCPS control. Hybrid cSAPs up-regulated gene expressions of focal adhesion kinase (FAK) and chondrocytes (AGG and SOX9) under static culture, while they also up-regulated osteocytes (COL1 and RUNX2) under dynamic culture. THP-1 macrophages were at M0 state on all cSAPs under static culture. However, cells became sensitive under dynamic culture. For example, some M1 genes (i.e., IL6, CD68, and TNFα) and M2 genes (i.e., IL10 and CD206) were down-regulated, while other M1 genes (i.e., IL1ß) and M2 genes (i.e., TGF-ß and IL1ra) were up-regulated, depending on the particle combinations. In conclusion, new hybrid cSAPs with thermoresponsive surface properties are versatile materials for stem cells and macrophages manipulation.

Harnessing Colloidal Self-Assembled Patterns (cSAPs) to Regulate Bacterial and Human Stem Cell Response at Biointerfaces In Vitro and In Vivo.

The generation of complex physicochemical signals on the surface of biomedical materials is still challenging despite the fact that a broad range of surface modification methods have been developed over the last few decades. Colloidal self-assembled patterns (cSAPs) are combinations of unique colloids differing in size and surface chemistry acting as building blocks that can be programmed to generate surface patterns with exquisite control of complexity. This study reports on producing a variety of pre-modified colloids for the fabrication of cSAPs as well as post-assembly modifications to yield complex surfaces. The surface of cSAPs presents hierarchical micro- and nanostructures, localized hydrophilic/hydrophobic characteristics, and tunable surface functionality imparted by the individual colloids. The selected cSAPs can control bacterial adhesion (S. aureus, P. aeruginosa, and E. coli) and affect the cell cycle of human bone marrow stem cells (hBMSCs). Moreover, in a mouse subcutaneous model, cSAPs with selective [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium (SBMA) modification can reduce the inflammatory response after being challenged with bacteria. This study reveals that functionalized cSAPs are versatile tools for controlling cellular responses at biointerfaces, which is instructive for biomaterials or biodevices.

Human Platelet Lysate Supports Mouse Skeletal Myoblast Growth but Suppresses Cell Fusion on Nanogrooves.

The fusion of skeletal myoblasts is a critical step in myotube formation and muscle maturation. Previously, we demonstrated that nanogrooves improved myoblast fusion via end-to-end fusion, which in turn generated parallel myotubes. However, the effect of serum components on the end-to-end fusion mechanism is unclear. In the current study, the synergistic effect of nanogrooves and human platelet lysate (hPL) on the growth and fusion of skeletal myoblasts was studied. Four types of nanogrooves (400 and 800 nm width; 100 and 400 nm depth) were used. Cell spreading, growth, and differentiation were screened on these nanogrooves in media formulated with hPL or fetal bovine serum (FBS), along with the flat substrate as a control group. The results showed that the deeper nanogrooves induced better alignment of myoblasts. hPL-adapted skeletal myoblasts (i.e., myoblasts@hPL) showed a smaller cell size with a more elongated morphology than myoblasts@FBS. During cell growth, the expression of myogenic genes (Myf5, MyoD, and MyoG) in myoblasts@hPL was lower than in myoblasts@FBS. During cell differentiation, myoblasts@hPL also expressed a lower level of myogenic and myosin heavy chain (MHC) genes. MHC-positive myoblasts@hPL without myotubes were found on all surfaces. Myomaker, an essential myoblast fusion gene, was upregulated during growth but downregulated during differentiation in myoblasts@hPL. Fibronectin-coated surfaces facilitate cell spreading and growth but still cannot support myoblast@hPL fusion. The results implied that hPL either lacks promotor factors or contains inhibitors on mouse skeletal myoblast fusion. This study reveals the effect of biophysical and biochemical cues on myoblast fusion and their potential for muscle tissue engineering.

Decoration of Material Surfaces with Complex Physicochemical Signals for Biointerface Applications.

The surface properties of biomaterials are crucial at controlling biological interactions. Cells or tissues sense different stimuli from surfaces and respond accordingly. A number of studies have reported that fabricating complex stimuli-presenting surfaces is beneficial for mimicking and understanding the in vivo scenario where multi-physicochemical cues are present. Biological responses toward these surfaces could be either negative, such as immune responses, or positive, such as tissue regeneration. An ideal material surface should, therefore, be multifunctional, triggering the desired biological process and suppressing the unwanted side effects. The methods for material surface decoration can be very sophisticated, depending on the applications such as biosensors, medical devices, and/or implants. To date, decorating material surfaces with complex chemistries and topographies is still challenging, and methods are not straightforward. The majority of the methods require multiple steps and combinational approaches that include mask-based techniques, lithography, wet or dry etching, wet chemistry, or vapor-based coatings, among others. Although these methods have been established in the laboratory, easy-to-access and straightforward approaches need to be explored. This Review summarizes the state-of-the-art techniques for generating patterned multichemical and multitopographic signals on material surfaces, and the potential of having these surfaces in biointerface applications.

Transcriptome-wide N 6 -methyladenosine methylome profiling of porcine muscle and adipose tissues reveals a potential mechanism for transcriptional regulation and differential methylation pattern.

BACKGROUND: N 6 -methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and potential regulatory functions of reversible m6A methylation on mRNA have been revealed by mapping of m6A methylomes in several species. m6A modification in active gene regulation manifests itself as altered methylation profiles in a tissue-specific manner or in response to changing cellular or species living environment. However, up to date, there has no data on m6A porcine transcriptome-wide map and its potential biological roles in adipose deposition and muscle growth. METHODS: In this work, we used methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) technique to acquire the first ever m6A porcine transcriptome-wide map. Transcriptomes of muscle and adipose tissues from three different pig breeds, the wild boar, Landrace, and Rongchang pig, were used to generate these maps. RESULTS: Our findings show that there were 5,872 and 2,826 m6A peaks respectively, in the porcine muscle and adipose tissue transcriptomes. Stop codons, 3'-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors, suggestive of a relationship between m6A mRNA methylation and nuclear genome transcription. Some genes showed tissue- and breed-differential methylation, and have novel biological functions. We also found a relationship between the m6A methylation extent and the transcript level, suggesting a regulatory role for m6A in gene expression. CONCLUSION: This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.

Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation.

Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

Cloning of porcine GPIHBP1 gene and its tissue expression pattern and genetic effect on adipose traits.

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism and is transported by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) from the interstitial spaces to the capillary lumen. Here, we cloned a cDNA and the genomic locus of the porcine GPIHBP1 gene, and investigated its tissue expression pattern and its genetic effects on adipose traits. Porcine GPIHBP1 exhibits a four-exon/three-intron structure, including a 543bp open reading frame that encodes 180 amino acids. The porcine GPIHBP1 protein shows 49%-65% homology and shares the major conserved structural domains of GPIHBP1 proteins in other mammals. Porcine GPIHBP1 mRNA levels were high in the adipose tissue, muscle and lung, and higher mRNA levels were observed in sows compared to boars in adipose tissues of the inner and outer layers of subcutaneous fat, abdominal fat, and suet fat. The mRNA expression pattern of porcine GPIHBP1 and LPL genes was similar in most tissues except for the lung. Thirty six single nucleotide polymorphisms (SNPs) were found in the porcine GPIHBP1 gene. Association analyses showed that the g.-255G>C and g.-626T>G SNPs are associated with intramuscular fat content, and that the g.-1557T>C and g.-1948G>A SNPs are associated with back fat thickness. In conclusion, porcine GPIHBP1 mRNA is abundantly expressed in the adipose tissue, muscle and lung, and gender affects GPIHBP1 mRNA expression levels; furthermore, four GPIHBP1 SNPs are genetic factors affecting adipose traits.

Comparison of Tripterygium wilfordii Hook F versus sulfasalazine in the treatment of rheumatoid arthritis: a randomized trial.

BACKGROUND: Extracts of the medicinal plant Tripterygium wilfordii Hook F (TwHF) have been used in China for centuries to treat a spectrum of inflammatory diseases. OBJECTIVE: To compare the benefits and side effects of TwHF extract with those of sulfasalazine for the treatment of active rheumatoid arthritis. DESIGN: Randomized, controlled trial. A computer-generated code with random, permuted blocks was used to assign treatment. SETTING: 2 U.S. academic centers (National Institutes of Health, Bethesda, Maryland, and University of Texas, Dallas, Texas) and 9 rheumatology subspecialty clinics (in Dallas and Austin, Texas; Tampa and Fort Lauderdale, Florida; Arlington, Virginia; Duncanville, Pennsylvania; Wheaton and Greenbelt, Maryland; and Lansing, Michigan). PATIENTS: 121 patients with active rheumatoid arthritis and 6 or more painful and swollen joints. INTERVENTION: TwHF extract, 60 mg 3 times daily, or sulfasalazine, 1 g twice daily. Patients could continue stable doses of oral prednisone or nonsteroidal anti-inflammatory drugs but had to stop taking disease-modifying antirheumatic drugs at least 28 days before randomization. MEASUREMENTS: The primary outcome was the rate of achievement of 20% improvement in the American College of Rheumatology criteria (ACR 20) at 24 weeks. Secondary end points were safety; radiographic scores of joint damage; and serum levels of interleukin-6, cholesterol, cortisol, and adrenocorticotropic hormone. RESULTS: Outcome data were available for only 62 patients at 24 weeks. In a mixed-model analysis that imputed data for patients who dropped out, 65.0% (95% CI, 51.6% to 76.9%) of the TwHF group and 32.8% (CI, 21.3% to 46.0%) of the sulfasalazine group met the ACR 20 response criteria (P=0.001). Patients receiving TwHF also had significantly higher response rates for ACR 50 and ACR 70 in mixed-model analyses. Analyses of only completers showed similar significant differences between the treatment groups. Significant improvement was demonstrated in all individual components of the ACR response, including the Health Assessment Questionnaire disability score. Interleukin-6 levels rapidly and significantly decreased in the TwHF group. Although not statistically significant, radiographic progression was lower in the TwHF group. The frequency of adverse events was similar in both groups. LIMITATIONS: Only 62% and 41% of patients continued receiving TwHF extract and sulfasalazine, respectively, during the 24 weeks of the study. Long-term outcome data were not collected on participants who discontinued treatment. CONCLUSION: In patients who continued treatment for 24 weeks and could also use stable oral prednisone and nonsteroidal anti-inflammatory drugs, attainment of the ACR 20 response criteria was significantly greater with TwHF extract than with sulfasalazine.

Effective therapy for nephritis in (NZB x NZW)F1 mice with triptolide and tripdiolide, the principal active components of the Chinese herbal remedy Tripterygium wilfordii Hook F.

OBJECTIVE: Triptolide and tripdiolide are thought to be active components of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F, which has been shown to be effective in treating murine lupus nephritis. This study was undertaken to examine the therapeutic effect of triptolide and tripdiolide on established lupus nephritis in (NZB x NZW)F1 mice. METHODS: (NZB x NZW)F1 mice were treated with vehicle, triptolide, or tripdiolide for 15 weeks beginning at the age of 29 weeks (after the development of lupus nephritis). Body weight, proteinuria, and anti-double-stranded DNA (anti-dsDNA) antibodies were monitored, and the kidney and spleen were assessed histologically. Culture supernatants of spleen mononuclear cells were assayed for cytokines. RESULTS: By 28 weeks, most (NZB x NZW)F1 mice had developed lupus nephritis. Vehicle-treated mice exhibited progressive proteinuria, hypoalbuminemia, elevated blood urea nitrogen (BUN) levels, and evidence of severe nephritis. In contrast, proteinuria and BUN levels were significantly reduced in mice treated with either triptolide or tripdiolide as compared with those treated with vehicle. There was no hypoalbuminemia or apparent evidence of lupus nephritis in mice treated with either of the 2 diterpenoids. At 44 weeks of age, the survival rate in mice treated with vehicle (35.7%) was markedly lower than that in mice treated with either triptolide (87.5%) or tripdiolide (88.2%). The mean level of anti-dsDNA antibody in mice treated with tripdiolide was lower than that in the vehicle-treated mice upon completion of the treatment course. Production of tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein 1 by spleen cells was also decreased after diterpenoid therapy. CONCLUSION: Therapy with triptolide or tripdiolide significantly ameliorated lupus nephritis in (NZB x NZW)F1 mice, reduced cytokine and chemokine production, and prolonged survival.

Therapeutic impact of the ethyl acetate extract of Tripterygium wilfordii Hook F on nephritis in NZB/W F1 mice.

This study was designed to examine the potential use of the ethyl acetate (EA) extract of Tripterygium wilfordii Hook F (TwHF), a Chinese herbal medicine, in the treatment of systemic lupus erythematosus. A total of 48 28-week-old female NZB/W F1 mice were randomly divided into three groups and orally administered vehicle or the EA extract of TwHF at 18.25 mg/kg (EAlow) or 36.5 mg/kg (EAhigh) for 14 weeks. Proteinuria and serum anti-double-stranded (ds)DNA antibody titers were assayed before and after treatment. At the end of treatment, all animals were sacrificed and pathological changes in the kidneys were examined by observers blinded to the treatment regimens. Immunohistological studies were carried out on kidneys and spleens. At 28 weeks of age, proteinuria (>30 mg/dl) and anti-dsDNA antibodies were found in all mice in the three groups. Fourteen, sixteen and fifteen mice in the vehicle, EAlow and EAhigh groups, respectively, completed at least four weeks of treatment. At the end of treatment, the mean proteinuria of the EAlow and EAhigh groups was significantly less than that of the vehicle group and no different from proteinuria at the onset of treatment. Histological evidence of glomerulonephritis, glomerular deposition of IgG and complement 3 and cellular infiltration in the interstitium and perivascular regions were significantly less severe in the EA extract treated mice than in vehicle treated mice. Treatment with the EA extract significantly inhibited the progression of kidney disease in NZB/W F1 mice, though had no significant effect on the levels of anti-dsDNA antibody.

Benefit of an extract of Tripterygium Wilfordii Hook F in patients with rheumatoid arthritis: a double-blind, placebo-controlled study.

OBJECTIVE: To examine the safety and efficacy of an extract of Tripterygium wilfordii Hook F (TWHF) in the treatment of patients with rheumatoid arthritis (RA). METHODS: An ethanol/ethyl acetate extract from the roots of TWHF was prepared and used in a prospective, double-blind, placebo-controlled study in patients with longstanding RA in whom conventional therapy had failed. Patients were randomly assigned to receive either placebo or low-dose (180 mg/day) or high-dose (360 mg/day) extract for 20 weeks, followed by an open-label extension period. Clinical responses were defined as 20% improvement in disease activity according to the American College of Rheumatology criteria. Side effects were actively queried and recorded at each visit. RESULTS: A total of 35 patients were enrolled in the trial; 21 patients completed the 20-week study. One patient from each group withdrew because of side effects. Twelve, 10, and 10 patients in the placebo, low-dose, and high-dose groups, respectively, completed at least 4 weeks of treatment. Of these patients, 8 and 4 in the high-dose and low-dose groups, but none in the placebo group, met criteria for clinical response. Four, 4, and 7 patients in the placebo, low-dose, and high-dose groups, respectively, were enrolled in the open-label extension; of these, 2, 4, and 5 patients, respectively, met criteria for clinical response. The most common side effect was diarrhea, which caused 1 patient in the high-dose group to withdraw from the trial. No patients withdrew because of adverse events during the open-label extension. CONCLUSION: The ethanol/ethyl acetate extract of TWHF shows therapeutic benefit in patients with treatment-refractory RA. At therapeutic dosages, the TWHF extract was well tolerated by most patients in this study.