Botanical preparation HX109 inhibits macrophage-mediated activation of prostate epithelial cells through the CCL4-STAT3 pathway: implication for the mechanism underlying HX109 suppression of prostate hyperplasia.

Benign prostatic hyperplasia (BPH) is one of the most frequently observed diseases in the elderly male population worldwide. A variety of factors such as aging, hormonal imbalance, chronic inflammation, and oxidative stress play an important role in its pathogenesis. We have previously shown that HX109, an ethanol extract prepared from 3 plants (Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera), alleviates prostate hyperplasia in the BPH rat model and suppresses AR signaling by upregulating Ca2+/CAMKKß and ATF3. In this study, we used macrophage cell lines to examine the effects of HX109 on inflammation, which is considered an important causative factor in BPH pathogenesis. In the co-culture system involving macrophage-prostate epithelial cells, HX109 inhibited macrophage-induced cell proliferation, migration and epithelial-mesenchymal transition (EMT) by inhibiting the expression of CCL4 and the phosphorylation of STAT3. Furthermore, HX109 inhibited the expression of inflammatory cytokines and the phosphorylation of p65 NF-κB in a concentration dependent manner. Taken together, our results suggested that HX109 could regulate macrophage activation and its crosstalk with prostate cells, thereby inhibiting BPH.

PG102 Upregulates IL-37 through p38, ERK, and Smad3 Pathways in HaCaT Keratinocytes.

IL-37 is an immunomodulatory cytokine that suppresses inflammation in various cell types and disease models. However, its role in keratinocytes has not been clearly understood, and there has been no report on the agents that can increase the expression of IL-37 in keratinocytes. In this study, we investigated the effects of silencing IL37 in HaCaT keratinocytes and the molecular mechanisms involved in the upregulation of IL-37 by PG102, a water-soluble extract from Actinidia arguta. It was found that knockdown of IL37 resulted in the augmented expression of antimicrobial peptides (AMPs) in response to cytokine stimulation. PG102 increased the expression of IL-37 at both mRNA and protein levels presumably by enhancing the phosphorylation of Smad3, ERK, and p38. Indeed, when cells were treated with specific inhibitors for these signaling molecules, the expression level of IL-37 was reduced. PG102 also promoted colocalization of phospho-Smad3 and IL-37. Our results suggest that IL-37 inhibits the expression of AMPs and that PG102 upregulates IL-37 through p38, ERK, and Smad3 pathways in HaCaT cells.

Botanical Formulation HX109 Ameliorates TP-Induced Benign Prostate Hyperplasia in Rat Model and Inhibits Androgen Receptor Signaling by Upregulating Ca2+/CaMKKß and ATF3 in LNCaP Cells.

Benign prostatic hyperplasia (BPH) is a common disease in the elderly male population throughout the world. Among other factors, androgen dysregulation has been known to play major roles in its pathogenesis. HX109 is a botanical formulation prepared from a mixture of Taraxacum officinale, Cuscuta australis, and Nelumbo nucifera, which have traditionally been used-usually along with other plants-to treat urinary diseases. An ethanol extract was prepared from a mixture of these three plants, and its quality was controlled through cell-based bioassays and by quantification of several marker compounds by high-performance liquid chromatography (HPLC). In the testosterone propionate (TP)-induced prostate hyperplasia rat model, oral administration of HX109 ameliorated prostate enlargement and histological changes induced by TP. In LNCaP cells, a human prostate epithelial cell line, HX109 repressed AR-mediated cell proliferation and the induction of androgen receptor (AR) target genes at the transcriptional level without affecting the translocation or expression of AR. Such effects of HX109 on AR signaling were mediated through the control of activating transcriptional factor 3 (ATF3) expression, phosphorylation of calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß), and increases in intracellular calcium, as evidenced by data from experiments involving ATF3-specific siRNA, CaMKKß inhibitor, and calcium chelator, respectively. Taken together, our data suggest that HX109 might be used as a starting point for developing therapeutic agents for the treatment of BPH.

A Water-Soluble Extract from Actinidia arguta Ameliorates Psoriasis-Like Skin Inflammation in Mice by Inhibition of Neutrophil Infiltration.

Psoriasis is a chronic inflammatory disease with complex etiology involving multiple factors. Current treatment methods are highly limited and there is a strong need for the development of safer and efficacious agents. We have previously shown that a water-soluble extract derived from hardy kiwifruit Actinidia arguta, called PG102, shows potent anti-inflammatory effects. Based on its reported biological activities, the effects of PG102 were examined on imiquimod-induced psoriasis-like skin inflammation. Our results showed that topical application of PG102 ameliorates clinical symptoms of psoriasis, reducing skin thickness and Interleukin (IL)-17A level in draining lymph nodes without causing any adverse effects. Treatment with PG102 on cytokine-stimulated HaCaT cells suppressed hyperproliferation and downregulated the expression of various chemokines and antimicrobial peptides known to induce neutrophil infiltration. These anti-inflammatory activities of PG102 were mediated via inhibition of NF-κB and signal transducer of activation (STAT) signaling. We also found decreased neutrophil chemotaxis both in vitro and in vivo. Taken together, PG102 has potential as a safe and effective reagent for the treatment of psoriasis.

Dehydrodiconiferyl Alcohol Inhibits Osteoclast Differentiation and Ovariectomy-Induced Bone Loss through Acting as an Estrogen Receptor Agonist.

Estrogen deficiency after menopause increases bone loss by activating RANKL-induced osteoclast differentiation. Dehydrodiconiferyl alcohol (DHCA), a lignan originally isolated from Cucurbita moschata, has been thought to be a phytoestrogen based on its structure. In this study, we tested whether DHCA could affect RANKL-induced osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo. In RAW264.7 cells, DHCA inhibited RANKL-induced differentiation of osteoclasts. Consistently, expression of the six osteoclastogenic genes induced by RANKL was down-regulated. DHCA was also shown to suppress the NF-κB and p38 MAPK signaling pathways by activating AMPK. Data from transient transfection assays suggested that DHCA might activate the estrogen receptor signaling pathway. Effects of DHCA on RANKL-induced osteoclastogenesis were reduced when cells were treated with specific siRNA to ERα, but not to ERß. Interestingly, DHCA was predicted from molecular docking simulation to bind to both ERα and ERß. Indeed, data from an estrogen receptor competition assay revealed that DHCA acted as an agonist on both estrogen receptors. In the ovariectomized (Ovx) mouse model, DHCA prevented Ovx-induced bone loss by inhibiting osteoclastogenesis. Taken together, our results suggest that DHCA may be developed as an efficient therapeutic for osteoporosis by regulating osteoclastogenesis through its estrogenic effects.

PG201 protects mice from experimental autoimmune encephalomyelitis via suppression of effector T cell activation.

BACKGROUND: PG201 is a botanical formulation, approved as an ethical drug (ETC) phytomedicine for treatment of patients with osteoarthritis in Korea, following satisfactory phase II and phase III studies. This phytomedicine was previously been shown to possess significant anti-inflammatory activities, presumably via the control of Th1 and Th17 cells in animal models and in vitro cell culture systems. PURPOSE: In this study, the possibility of using PG201 to treat multiple sclerosis was explored. METHODS: In vitro, the effect of PG201 on the differentiation of CD4+ T cells was investigated. To test the effects of PG201 in vivo, a mouse experimental autoimmune encephalomyelitis (EAE) model was used. RESULTS: It was found that PG201 treatment decreased the frequency of both CD4+T-bet+ and CD4+RORγt+T cells. In addition, the production of interferon- gamma (IFN-γ) and interleukin-17 (IL-17) from respective Th cells was highly reduced. The data from western blots showed that the amount of phosphorylated c-Jun, but not that of p65, was decreased by PG201. Consistently, the level of luciferase activity was downregulated by PG201 in activator protein 1 (AP-1) reporter plasmid assays. In mice pretreated with PG201, the day of onset was delayed and clinical symptoms of EAE were significantly improved in a dose-dependent manner. Consistent with these results, the number of infiltrated cells and the expression level of pro-inflammatory molecules were decreased. CONCLUSION: These findings indicate that PG201 may exert strong immunomodulatory effects in the EAE model via suppression of T cell activation, and that PG201 is a therapeutic reagent for the treatment of multiple sclerosis.

Dehydrodiconiferyl alcohol promotes BMP-2-induced osteoblastogenesis through its agonistic effects on estrogen receptor.

Estrogen deficiency results in an imbalance between the levels of bone-resorping osteoclasts and bone-forming osteoblasts, eventually leading to overall bone loss. Dehydrodiconiferyl alcohol (DHCA), a lignan compound originally isolated from Cucurbita moschata, has been shown to bind to estrogen receptor, and indeed exhibits various activities of estrogen, such as anti-inflammatory and anti-oxidative stress effects. In this study, we tested whether synthetic DHCA could affect the BMP-2-induced osteoblastogenesis in vitro. In MC3T3-E1 cells, DHCA promoted BMP-2-induced differentiation of osteoblasts. Consistently, the expression of three osteoblastogenic genes known to be induced by BMP-2, ALP, osteocalcin and OPG, was up-regulated by DHCA treatment. DHCA was also shown to activate the production of RUNX2 by activating Smad1/5/9 and AMPK. Data from transient transfection assays suggested that DHCA might activate the estrogen receptor signaling pathway. Effects of DHCA on BMP-2-induced osteoblastogenesis were reduced when cells were treated with a specific siRNA to ERα or ERß. Taken together, our results suggest that DHCA may be developed as an efficient therapeutic for osteoporosis by regulating osteoblastogenesis through its estrogenic effects.

Water soluble extracts from Actinidia arguta, PG102, attenuates house dust mite-induced murine atopic dermatitis by inhibiting the mTOR pathway with Treg generation.

ETHNOPHARMACOLOGICAL RELEVANCE: Actinidia arguta is widespread in northeastern Asia, being found in Siberia, Korea, Japan, and northern China. These fruits have been documented to regulate the uncontrolled heat of body resulting in various allergic diseases in the Korean traditional medicine. PG102, a water-soluble extract from an edible fruit, A. arguta, has been previously shown to control various factors involved in allergic pathogenesis. AIM OF THE STUDY: In this study, we investigated whether PG102 prevents chronic allergic reactions via the generation of Tregs, which play a preventive role in the pathogenesis of allergic disease. METHODS AND RESULTS: In dust mite extract-induced chronic atopic dermatitis, orally administered PG102 inhibited symptoms of dermatitis, including ear swelling and erythema, and decreased lymphocyte infiltration into the inflamed region. Moreover, PG102 reduced inflammatory T cell responses and increased the expression levels of Foxp3 and other Treg-related genes. PG102 treatment enhanced the induction of CD4+Foxp3+ Tregs from naive CD4+CD62L+ T cells, probably via the inhibition of mTOR activation and the phosphorylation of STAT5 rather than using the TGF-ß signaling pathway. CONCLUSION: PG102 may have potential as an orally active immunosuppressor for preventing chronic inflammatory diseases.

Effective suppression of nitric oxide production by HX106N through transcriptional control of heme oxygenase-1.

Heme oxygenase-1 (HO-1) has been suggested to be a key neuroprotective enzyme because of its widespread distribution in the brain as well as its strong antioxidative effects. HX106N, a water-soluble botanical formulation, has previously been demonstrated to prevent amyloid ß-induced memory impairment and oxidative stress in mice by upregulating HO-1 levels. In this study, the underlying molecular mechanisms of HX106N-induced HO-1 expression were investigated using BV-2 cells, a murine microglial cell line, and primary microglia. Treatment with HX106N induced the expression of HO-1 at the transcriptional level through the stress-responsive element-containing enhancer present in the ho-1 promoter. Nuclear factor E2-related factor 2 (Nrf2) was activated in cells treated with HX106N. The results from knockdown assay showed that small interfering RNA of Nrf2 attenuated HX106N-mediated HO-1 expression. Pharmacological inhibitors of p38 and JNK mitogen-activated protein kinases suppressed the HX106N-mediated induction of HO-1. The NF-κB signaling pathway was activated by HX106N and played a role in HX106N-induced HO-1 expression. Furthermore, HO-1 and one of its by-products during the enzymatic degradation of heme, CO, were found to be involved in HX106N-mediated suppression of NO production. Taken together, these data indicate that HX106N exerts potent antioxidative effects by increasing the expression of HO-1 through multiple signaling pathways, leading to the suppression of NO production.