Chinese Herbal Formula Huayu-Qiangshen-Tongbi Decoction Attenuates Rheumatoid Arthritis through Upregulating miR-125b to Suppress NF-κB-Induced Inflammation by Targeting CK2.

The Huayu-Qiangshen-Tongbi (HQT) decoction, a Chinese medical formula, has been identified to show a potent therapeutic effect on rheumatoid arthritis (RA). However, the specific molecular mechanism of HQT in RA has not been well studied. In the present study, LPS-treated human rheumatoid fibroblast-like synoviocyte (FLS) MH7A cells and collagen-induced arthritis (CIA) mice were utilized as in vitro and in vivo models. Our results demonstrated that HQT could efficiently inhibit RA-induced inflammation by reducing the production of cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). Moreover, HQT significantly upregulated the expression of miR-125b. Besides, analysis of bioinformatics suggested casein kinase 2 (CK2) was a potential target of miR-125b. Luciferase reporter assay was performed and revealed that miR-125b suppressed CK2 expression in MH7A cells. Furthermore, miR-125b inhibited LPS-induced NF-kappa-B (NF-κB) activation, which is a downstream target of CK2. In addition, the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) and NF-kappa-B inhibitor alpha (IkB-α) enhanced the inhibitory effect of miR-125b on the expression of TNF-α, IL-1ß, and IL-6. Taken together, our study revealed that HQT could attenuate RA through upregulating miR-125b to suppress NF-κB-induced inflammation by targeting CK2. The findings of this study should facilitate investigating the mechanism of HQT on RA and discovering novel therapeutic targets for RA.

Sustained Drug Treatment Alters the Gut Microbiota in Rheumatoid Arthritis.

Several studies have investigated the causative role of the microbiome in the development of rheumatoid arthritis (RA), but changes in the gut microbiome in RA patients during drug treatment have been less well studied. Here, we tracked the longitudinal changes in gut bacteria in 22 RA patients who were randomized into two groups and treated with Huayu-Qiangshen-Tongbi formula (HQT) plus methotrexate (MTX) or leflunomide (LEF) plus MTX. There were differences in the gut microbiome between untreated (at baseline) RA patients and healthy controls, with 37 species being more abundant in the RA patients and 21 species (including Clostridium celatum) being less abundant. Regarding the functional analysis, vitamin K2 biosynthesis was associated with RA-enriched bacteria. Additionally, in RA patients, alterations in gut microbial species appeared to be associated with RA-related clinical indicators through changing various gut microbiome functional pathways. The clinical efficacy of the two treatments was further observed to be similar, but the response trends of RA-related clinical indices in the two treatment groups differed. For example, HQT treatment affected the erythrocyte sedimentation rate (ESR), while LEF treatment affected the C-reactive protein (CRP) level. Further, 11 species and 9 metabolic pathways significantly changed over time in the HQT group (including C. celatum, which increased), while only 4 species and 2 metabolic pathways significantly changed over time in the LEF group. In summary, we studied the alterations in the gut microbiome of RA patients being treated with HQT or LEF. The results provide useful information on the role of the gut microbiota in the pathogenesis of RA, and they also provide potentially effective directions for developing new RA treatments.

The mechanism of Chinese herbal formula HQT in the treatment of rheumatoid arthritis is related to its regulation of lncRNA uc.477 and miR-19b.

Rheumatoid arthritis (RA) pathogenesis has been associated with dysregulation of long noncoding RNA (lncRNA) and microRNA (miRNA) expression in serum and in lesioned tissue. In this study, a microarray assay was performed to study the profile of lncRNAs in the serum of RA patients and healthy donors, and a set of novel lncRNAs associated with RA was identified. For the remainder of the study, focus is on the top hit, lncRNA uc.477. The upregulation of lncRNA uc.477 and downregulation of miR-19b were validated in the serum of RA patients compared to that of healthy donors, and similar results were further confirmed by quantitative real-time PCR analysis of a cell line: RA-derived human fibroblast-like synoviocytes (HFLS-RA). LncRNA uc.477 could interfere with the processing of pri-miR-19b to produce its mature form and thereby played a pro-inflammatory role. In addition, Huayu Qiangshen Tongbi formula (HQT), a traditional Chinese medicine (TCM), has been shown to exert a promising therapeutic effect on RA and to exhibit long-term safety in our previous clinical retrospective study. Importantly, HQT treatment normalized the levels of lncRNA uc.477 and miR-19b in HFLS-RA in vitro and in mouse models of collagen-induced arthritis. HQT treatment, knockdown of lncRNA uc.477, and overexpression of miR-19b resulted in a comparable inhibition of pro-inflammatory cytokine gene expression in HFLS-RA cells. Together, these data suggest that the therapeutic effects of HQT on RA are closely related to its modulation of lncRNA uc.477 and miR-19b.

Paeonol attenuates inflammation by targeting HMGB1 through upregulating miR-339-5p.

Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Paeonol, an active ingredient in moutan cortex (a Chinese herb), has many pharmacological activities, such as anti-inflammatory and antitumour actions. Previous studies have indicated that paeonol inhibits the expression of HMGB1 and the transcriptional activity of NF-κB. However, its underlying mechanism is still unknown. In this study, microarray assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Dual-luciferase assays indicated that miR-339-5p interacted with the 3' untranslated region (3'-UTR) of HMGB1. Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR-339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1ß and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-ß is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-ß expression. Positive mutual feedback between HMGB1 and IKK-ß was observed when we silenced HMGB1 or IKK-ß. These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-ß by upregulating miR-339-5p expression. In addition, we constructed CLP model mice by cecal ligation and puncture. Paeonol was used to intervene to investigate its anti-inflammatory effect in vivo. The results showed that paeonol could improve the survival rate of sepsis mice and protect the kidney of sepsis mice.

Anti-Inflammatory Effects of Shenfu Injection against Acute Lung Injury through Inhibiting HMGB1-NF-κB Pathway in a Rat Model of Endotoxin Shock.

Shenfu injection (SFI), a Chinese herbal medicine with substances extracted from Ginseng Radix et Rhizoma Rubra and Aconiti Lateralis Radix Praeparata, is widely used as an anti-inflammatory reagent to treat endotoxin shock in China. However, the mechanism of SFI in endotoxin shock remains to be illuminated. High mobility group box 1 (HMGB1), a vital inflammatory factor in the late stage of endotoxin shock, may stimulate multiple signalling cascades, including κB (NF-κB), a nuclear transcription factor, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, among others in the overexpression of downstream proinflammatory cytokines. An investigation into the effects of SFI on the inhibition of the HMGB1-NF-κB pathway revealed the contribution of SFI to acute lung injury (ALI) in a rat model of endotoxin shock. To assess the anti-inflammatory activity of SFI, 5 ml/kg, 10 ml/kg, or 15 ml/kg of SFI was administered to different groups of rats following an injection of LPS, and the mean arterial pressure (MAP) at 5 h and the survival rate at 72 h were measured. 24 h after LPS injection, we observed pathological changes in the lung tissue and measured the mRNA expression, production, translocation, and secretion of HMGB1, as well as the expression of the NF-κB signal pathway-related proteins inhibitor of NF-κB (IκB)-α, P50, and P65. We also evaluated the regulation of SFI on the secretion of inflammatory factors including interleukin-1 beta (IL-1ß) and TNF-α. SFI effectively prevented the drop in MAP, relieved lung tissue damage, and increased the survival rate in the endotoxin shock model in dose-dependent manner. SFI inhibited the transcription, expression, translocation, and secretion of HMGB1, increased the expression of toll-like receptor (TLR4), increased the production of IκB-α, and decreased the levels of P65, P50, and TNF-α in the lung tissue of endotoxin shock rats in a dose-dependent manner. Furthermore, SFI decreased the secretion of proinflammatory cytokines TNF-α and IL-1ß. In summary, SFI improves the survival rate of endotoxin shock, perhaps through inhibiting the HMGB1-NF-κB pathway and thus preventing cytokine storm.

Paeonol attenuates acute lung injury by inhibiting HMGB1 in lipopolysaccharide-induced shock rats.

High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding nuclear protein that facilitates gene transcription and the DNA repair response. However, HMGB1 may be released by necrotic cells as well as activated monocytes and macrophages following stimulation with lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), or tumor necrosis factor-α (TNF-α). Extracellular HMGB1 plays a critical role in the pathogenesis of acute lung injury (ALI) through activating the nuclear transcription factor κB (NF-κB) P65 pathway, thus, it may be a promising therapeutic target in shock-induced ALI. Paeonol (Pae) is the main active component of Paeonia suffruticosa, which has been used to inhibit the inflammatory response in traditional Chinese medicine. We have proven that Pae inhibits the expression, relocation and secretion of HMGB1 in vitro. However, the role of Pae in the HMGB1-NF-κB pathway remains unknown. We herein investigated the role of Pae in LPS-induced ALI rats. In this study, LPS induced a marked decrease in the mean arterial pressure (MAP) and survival rate (only 25% after 72 h), and induced severe pathological changes in the lung tissue of rats, which was accompanied by elevated expression of HMGB1 and its downstream protein NF-κB P65. Treatment with Pae significantly improved the survival rate (>60%) and MAP, and attenuated the pathological damage to the lung tissue in ALI rats. Western blotting revealed that Pae also inhibited the total expression of HMGB1, NF-κB P65 and TNF-α in the lung tissue of ALI rats. Moreover, Pae increased the expression of HMGB1 in the nucleus, inhibited the production of HMGB1 in the cytoplasm, and decreased the expression of P65 both in the nucleus and cytoplasm of lung tissue cells in LPS-induced ALI rats. The results were in agreement with those observed in the in vitro experiment. These findings indicate that Pae may be a potential treatment for ALI through its repression of the HMGB1-NF-κB P65 signaling pathway.