Pharmacokinetics, anti-rheumatoid arthritis activity, and active ingredient contents of Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. is a deciduous tree which contains various chemical ingredients. The main objective was to document the active chemical ingredients of Eucommia ulmoides Oliv. and their metabolic profiles in vivo, with a view to providing an experimental and theoretical basis for clarifying the mechanisms underlying the pharmacological activity of Eucommia ulmoides Oliv. against rheumatoid arthritis. Eight main active constituents of Eucommia ulmoides Oliv. bark (pinoresinol glucopyranoside, aucubin, geniposidic acid, geniposide, genipin, chlorogenic acid, quercetin and betulinic acid) were quantified using high-performance liquid chromatography (HPLC). This paper additionally identified and characterized prototype metabolites via ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with the Human Metabolome Database (HMDB) and literature comparisons. Ultra pressure liquid chromatography-mass spectrometry/ mass spectrometry (UPLC-MS/MS) was subsequently employed to quantify these components in blood over time and evaluate their pharmacokinetic characteristics. The anti-rheumatoid arthritis effects of genipin, pinoresinol glucopyranoside and their combinations were assessed using in vitro cellular assays. We identified and characterized a total of 53 ingredients from Eucommia ulmoides Oliv. bark and plasma samples, among which 20 were confirmed as prototype metabolites. Meanwhile, this paper derived and analyzed the metabolic cleavage pathway of 8 index ingredients. Six of these compounds displayed rapid entry into blood, with high plasma exposure and fast elimination rates. Data from the in vitro cellular assay showed that aucubin, pinoresinol glucopyranoside, genipin, and combinations of these compounds effectively inhibit MH7A cell proliferation, reduce NO release, and decrease inflammatory factor levels.

Iridoid from Eucommia ulmoides Oliv. Exerts Antiarthritis Effects by Inhibiting the JAK2/STAT3 Signaling Pathway In Vivo and In Vitro.

The purpose of this study was to investigate the anti-inflammatory effects of EU-Idd both in vivo and in vitro. In vivo, we used the collagen-induced arthritis (CIA) rat model to investigate the efficacy of EU-Idd on rheumatoid arthritis. Hematoxylin-eosin staining and Safranin O-fast green staining were used to evaluate the pathological status of the ankle joints in CIA rats. Micro-CT scanning was used to investigate bone erosion of the ankle joints. In vitro, the effect of EU-Idd on Th17 cell differentiation was identified by flow cytometry. TRAP staining was used to detect osteoclast cells. HFLS-RA model cells, induced by tumor necrosis factor-α(TNF-α), were used to evaluate the anti-inflammatory effects of EU-Idd while the levels of related inflammatory cytokines and JAK2/STAT3 proteins were detected by RT-qPCR and western blotting. EU-Idd alleviated joint inflammation in CIA rats and exerted protective effects on the ankle joints. EU-Idd also prevented the differentiation of CD4+ T cells into Th17 cells, reduced the number of osteoclasts, and improved the expression levels of bone metabolism-related proteins including OPG and RANKL. Moreover, EU-Idd inhibited the invasion and migration of HFLS-RA cells and downregulated the expression of related inflammatory cytokine genes and the protein expression levels of p-JAK2 and p-STAT3, both in vivo and in vitro. EU-Idd exerts anti-inflammatory and osteoprotective effects by regulating the JAK2/STAT3 pathway in rheumatoid arthritis. These results are beneficial to excavate new pharmaceutical ingredients for rheumatoid arthritis from iridoid.

[Effects of triterpenoid and iridoid of Eucommiae Cortex on collagen-induced arthritis in rats].

The ethyl acetate fraction of ethanol extract of Eucommiae Cortex can effectively inhibit joint inflammation and bone destruction in rats with collagen-induced arthritis(CIA) and has a potential therapeutic effect on rheumatoid arthritis. The triterpenoid(EU-Tid) and iridoid(EU-Idd) of Eucommiae Cortex are derivatives isolated from the ethyl acetate fraction of the ethanol extract of Eucommiae Cortex, and it is not clear whether they have inhibitory effects on joint inflammation and bone erosion in CIA rats. Therefore, based on the CIA model, the effects of EU-Tid, EU-Idd, and their combination(EU-TP) on arthritis in rats were observed, and the material basis of Eucommiae Cortex against arthritis was further clarified. The samples were collected two and four weeks after administration to observe the pathological changes in different stages of arthritis in CIA rats. For the rats in the model control group, with the prolongation of the disease course, the paw volume and arthritis score increased and histopathological lesions aggravated. Compared with the model control group, the drug administration groups showed reduced paw volumes and arthritis scores, and improved joint lesions and cartilage destruction. Additionally, the mRNA expression levels of tumor necrosis factor-α(TNF-α), interleukin-17(IL-17), and interleukin-23(IL-23) in the spleen were down-regulated in the drug administration groups. EU-TP and EU-Tid at concentrations of 160 and 320 µg·mL~(-1) could significantly inhibit the proliferation of human fibroblast-like synoviocytes-RA(HFLS-RA) and nitric oxide(NO) release in the supernatant of RAW264.7 cells induced by lipopolysaccharide(LPS) at the concentration range of 10-80 µg·mL~(-1) in vitro. EU-Idd had no effect on the proliferation of HFLS-RA but could reduce the NO release at concentrations of 40 and 80 µg·mL~(-1). The results indicated that the terpenoids of Eucommiae Cortex had great potential in the treatment of rheumatoid arthritis.

Anti-inflammatory effects of aucubin in cellular and animal models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is known that aucubin (AU) exerts anti-inflammatory activity, but its effects and mechanisms in RA are unclear. This study investigated the anti-inflammatory effects and mechanisms of AU in vivo and in vitro. Human fibroblast-like synoviocyte cells from patients with RA (HFLS-RA), RAW264.7 cells, and MC3T3-E1 cells were used to evaluate the effects of AU on migration, invasion, apoptosis, osteoclast differentiation and production. Immunofluorescence was used to observe nuclear translocation of nuclear factor (NF)-κB, the double luciferase reporter gene method was used to observe NF-κB-p65 activity in AU-treated MC3T3-E1 cells. RT-qPCR was used to measure expression of bone metabolism and inflammation-related genes, and western blot was used to measure bone metabolism and NF-κB protein expression levels. Collagen-induced arthritis (CIA) rat model was used for pharmacodynamics study. Arthritis indexes were measured in the ankle and knee, histological staining and Micro-computed tomography were performed on the ankle joints. Also, inflammatory factor gene expression and the levels of NF-κB-related proteins were detected as in vitro. AU effectively inhibited HFLS-RA cell migration and invasion, promoted apoptosis, and inhibited RAW264.7 cell differentiation into osteoclasts, as well as inhibited NF-κB-p65 activity in MC3T3-E1 cells. Notably, AU significantly reduced the gene expression levels of three cell-related inflammatory factors and bone metabolism factors, effectively inhibited the expression of p-Iκκα ß, p-IκBα, and p-p65 proteins. In vivo, AU relieved joint inflammation, reduced related inflammatory factors, and inhibited NF-κB signaling. It could be used to treat RA-related synovial inflammation and bone destruction through the NF-κB pathway.

Structural and Functional Modulation of Gut Microbiota by Jiangzhi Granules during the Amelioration of Nonalcoholic Fatty Liver Disease.

Recently, accumulating evidence revealed that nonalcoholic fatty liver disease (NAFLD) is highly associated with the dysbiosis of gut microbiota. Jiang Zhi Granule (JZG), which is composed of five widely used Chinese herbs, has shown hypolipidemic effect, while whether such effect is mediated by gut microbiota is still unclear. Here, we found that both low and high doses of JZG (LJZ and HJZ) could improve hepatic steatosis and function, as well as insulin resistance in NAFLD mice. 16S rRNA gene sequencing revealed that JZG treatment could reverse the dysbiosis of intestinal flora in NAFLD mice, exhibiting a dose-dependent effect. Notably, HJZ could significantly reduce the relative abundance of Desulfovibrionaceae, while increasing the relative abundance of such as S24_7 and Lachnospiraceae. PICRUSt analysis showed that HJZ could significantly alter the functional profile of gut microbiota, including the reduction of the lipopolysaccharide biosynthesis and sulfur metabolism pathway, which is verified by the decreased levels of fecal hydrogen sulfide (H2S) and serum lipopolysaccharide binding protein (LBP). In addition, hepatic mRNA sequencing further indicated that the HJZ group can regulate the peroxisome proliferator-activated receptor (PPAR) pathway and inflammatory signaling pathway, as validated by RT-PCR and Western blot. We also found that different doses of JZG may regulate lipid metabolism through differentiated pathways, as LJZ mainly through the promotion of hepatic lipid hydrolysis, while HJZ mainly through the improvement of hepatic lipid oxidation. Taken together, JZG could modulate gut dysbiosis with dose-effect, alleviate inflammation level, and regulate hepatic lipid metabolism, which may subsequently contribute to the improvement of NAFLD. Our study revealed the underlying mechanisms in the improvement of NAFLD by a Chinese herbal compound, providing future guidance for clinical usage.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Inhibition of Rheumatoid Arthritis Using Bark, Leaf, and Male Flower Extracts of Eucommia ulmoides.

Eucommia ulmoides Oliv., a native Chinese plant species, has been used as a traditional Chinese medicine formulation to treat rheumatoid arthritis (RA), strengthen bones and muscles, and lower blood pressure. Various parts of this plant such as the bark, leaves, and flowers have been found to have anti-inflammatory properties. E. ulmoides has potential applications as a therapeutic agent against bone disorders, which were investigated in this study. In vitro, RA joint fibroblast-like synoviocytes (RA-FLS) were treated with different concentrations (0, 25, 50, 100, 200, 400, 800, and 1000 µg/mL) of E. ulmoides bark, leaf, and male flower alcoholic extracts (EB, EL, and EF, respectively) to determine their potential cytotoxicity. Tumor necrosis factor- (TNF-) α and nitric oxide (NO) levels in RA-FLS were quantified using enzyme-linked immunosorbent assay (ELISA). Furthermore, collagen-induced arthritis (CIA) rats were treated with EB, EL, EF, Tripterygium wilfordii polyglycoside (TG) or the normal control (Nor), and then ankle joint pathology, bone morphology, and serum and spleen inflammatory cytokine levels were evaluated. The results showed that, in RA-FLS, EB, EL, and EF were not cytotoxic; EB and EF reduced TNF-α supernatant levels; and EB, EL, and EF reduced NO levels. The results of in vivo experiments showed that EB, EL, and EF alleviated ankle swelling and joint inflammation, while all extracts diminished inflammatory cell infiltration, pannus and bone destruction, and bone erosion. All tested extracts inhibited interleukin- (IL-) 6, IL-17, and TNF-α mRNA in the spleen of CIA rats, while EB most effectively reduced osteoclasts and inhibited bone erosion. EF showed the most obvious inhibition of inflammatory factors and pannus. Thus, EB, EL, and EF may alleviate bone destruction by inhibiting inflammation.

[Development status and research strategy of traditional Chinese medicine temporary prescription preparation technology].

Temporary prescription preparation is the preparation processed into different dosage forms by relevant pharmacist according to the temporary preparation requirement and the personalized prescription made by the doctor in accordance with the syndrome differentiation and drug performance.It is an important part in personalized pharmaceutical services.Rational design of process route,production equipment and quality control method for the temporary prescription preparation,and establishment of technology research strategy and mode in accordance with the characteristics of traditional Chinese medicine temporary prescription preparations play an important role in promoting the development of the temporary prescription preparations.To promote the normalization,standardization and intelligent development of temporary prescription preparations,we would comprehensively summarize the significance,policy,technology characteristics,technology research status quo and existing problems in this paper,and put forward the research direction of temporary prescription preparation technology based on the physical properties of raw materials,equipment research strategy,and intelligent manufacturing technology.Thus it will push the inheritance and innovation of temporary prescription preparation.

Anti-inflammatory effects of Eucommia ulmoides Oliv. male flower extract on lipopolysaccharide-induced inflammation.

BACKGROUND: Eucommia ulmoides Oliv. is a medicinal plant native to China, with its bark (Eucommiae Cortex) traditionally being used for medicinal purposes. Previous research has shown that Eucommia male flowers can exert anti-inflammatory, analgesic, antibacterial, and other pharmacological effects, including immune regulation. This study explored the anti-inflammatory effects of the 70% ethanol extract of male flowers (EF) of E. ulmoides in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-administered mice. METHODS: Cytotoxicity of EF for RAW 264.7 cells was investigated using Cell Counting Kit-8. The production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 was determined using enzyme-linked immunosorbent assays. IL-17, IL-23, and IL-10 mRNA levels were determined using quantitative real-time polymerase chain reaction. Activation of the nuclear factor (NF)-κB pathway in RAW 264.7 cells was investigated via Western blotting. In vivo anti-inflammatory effects of EF were studied in an LPS-induced acute inflammation mouse model by analyzing lung tissue histopathology, serum TNF-α and IL-6 levels, and myeloperoxidase (MPO) activity in lung tissue. RESULTS: EF showed no significant cytotoxicity at concentrations from 10 to 60 µg/mL (cell viability > 80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60 µg/mL, 120 µg/mL, and 250 µg/mL vs. negative control: 87.31 ±â€Š2.39% vs. 100.00 ±â€Š2.50%, P = 0.001; 79.01 ±â€Š2.56 vs. 100.00 ±â€Š2.50%, P < 0.001; and 64.83 ±â€Š2.50 vs. 100.00 ±â€Š2.50%, P < 0.001), suppressed NO (EF 20 µg/mL and 30 µg/mL vs. LPS only, 288.81 ±â€Š38.01 vs. 447.68 ±â€Š19.07 µmol/L, P = 0.004; and 158.80 ±â€Š45.14 vs. 447.68 ±â€Š19.07 µmol/L, P < 0.001), TNF-α (LPS+EF vs. LPS only, 210.20 ±â€Š13.85 vs. 577.70 ±â€Š5.35 pg/mL, P < 0.001), IL-1ß (LPS+EF vs. LPS only, 193.30 ±â€Š10.80 vs. 411.03 ±â€Š42.28 pg/mL, P < 0.001), and IL-6 (LPS+EF vs. LPS only, 149.67 ±â€Š11.60 vs. 524.80 ±â€Š6.24 pg/mL, P < 0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23 ±â€Š0.02 vs. 0.43 ±â€Š0.12, P < 0.001), IL-23 (LPS+EF vs. LPS only, 0.29 ±â€Š0.01 vs. 0.42 ±â€Š0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30 ±â€Š0.01 vs. 0.47 ±â€Š0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20 µg/mL and 30 µg/mL vs. LPS only: 0.78 ±â€Š0.06 vs. 1.17 ±â€Š0.08, P < 0.001; and 0.90 ±â€Š0.06 vs. 1.17 ±â€Š0.08, P =0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20 µg/mL and 30 µg/mL vs. LPS only: 0.25 ±â€Š0.01 vs. 0.63 ±â€Š0.03, P < 0.001; and 0.31 ±â€Š0.01 vs. 0.63 ±â€Š0.03, P < 0.001), LPS+EF 30 µg/mL inhibited IκB kinase (IKKα/ß) phosphorylation (LPS+EF 30 µg/mL vs. LPS only, 1.12 ±â€Š0.14 vs. 1.71 ±â€Š0.25, P = 0.002) in RAW 264.7 cells. Furthermore, EF 10 mg/kg and EF 20 mg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 199.99 ±â€Š186.49 vs. 527.90 ±â€Š263.93 pg/mL, P=0.001; and 260.56 ±â€Š175.83 vs. 527.90 ±â€Š263.93 pg/mL, P = 0.005), and IL-6 (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 41.26 ±â€Š30.42 vs. 79.45 ±â€Š14.16 pg/ ml, P = 0.011; and 42.01 ±â€Š26.26 vs. 79.45 ±â€Š14.16 pg/mL, P = 0.012) levels and MPO (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 3.19 ±â€Š1.78 vs. 5.39 ±â€Š1.51 U/g, P = 0.004; and 3.32 ±â€Š1.57 vs. 5.39 ±â€Š1.51 U/g, P = 0.006) activity in lung tissue. CONCLUSIONS: EF could effectively inhibit the expression of inflammatory factors and overactivation of neutrophils. Further investigation is needed to evaluate its potential for anti-inflammation therapy.

Comparative Studies of Different Extracts from Eucommia ulmoides Oliv. against Rheumatoid Arthritis in CIA Rats.

To compare efficacy of different extracts from Eucommia ulmoides Oliv. with both immune inflammation and joint destruction in collagen induced arthritis (CIA) rat model. Rats were divided into normal group (Nor), control group (CIA), TG group (treated with tripterygium glycoside), E70 group (treated with 70% ethanol extract from Eucommia ulmoides Oliv.), EA group (treated with ethyl acetate fraction from E70), and EN group (treated with n-butyl alcohol fraction from E70). All extracts from Eucommia ulmoides Oliv. could significantly inhibit ankle swelling, pathological manifestations, and cytokine levels in serum and spleen, by using foot volume measurement, H&E staining, ELISA, and RT-QPCR methods, respectively. All extracts could significantly inhibit rough joint surface and marginal osteophytes, improve RANKL/OPG ratio, and decrease MMP-9 expression, by using micro-CT and immunohistochemical staining. The activation of IKK/NF-κB signaling pathway was also inhibited by all extracts. In addition, ethyl acetate fraction from E70 presented better effect on RANKL/OPG system. This study identified effective extracts from Eucommia ulmoides Oliv. relieving immune inflammation and maintaining structural integrity of joints in CIA rats.

Pulse Wave Cycle Features Analysis of Different Blood Pressure Grades in the Elderly.

BACKGROUND AND OBJECTIVE: The same range of blood pressure values may reflect different vascular functions, especially in the elderly. Therefore, a single blood pressure value may not comprehensively reveal cardiovascular function. This study focused on identifying pulse wave features in the elderly that can be used to show functional differences when blood pressure values are in the same range. METHODS: First, pulse data were preprocessed and pulse cycles were segmented. Second, time domain, higher-order statistics, and energy features of wavelet packet decomposition coefficients were extracted. Finally, useful pulse wave features were evaluated using a feature selection and classifier design. RESULTS: A total of 6,075 pulse wave cycles were grouped into 3 types according to different blood pressure levels and each group was divided into 2 categories according to a history of hypertension. The classification accuracy of feature selection in the 3 groups was 97.91%, 95.24%, and 92.28%, respectively. CONCLUSION: Selected features could be appropriately used to analyze cardiovascular function in the elderly and can serve as the basis for research on a cardiovascular risk assessment model based on Traditional Chinese Medicine pulse diagnosis.

A strategy to identify and quantify closely related adulterant herbal materials by mass spectrometry-based partial least squares regression.

In this study, a new strategy combining mass spectrometric (MS) techniques with partial least squares regression (PLSR) was proposed to identify and quantify closely related adulterant herbal materials. This strategy involved preparation of adulterated samples, data acquisition and establishment of PLSR model. The approach was accurate, sensitive, durable and universal, and validation of the model was done by detecting the presence of Fritillaria Ussuriensis Bulbus in the adulteration of the bulbs of Fritillaria unibracteata. Herein, three different MS techniques, namely wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI/MS), ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and UPLC-triple quadrupole tandem mass spectrometry (UPLC-TQ/MS), were applied to obtain MS profiles for establishing PLSR models. All three models afforded good linearity and good accuracy of prediction, with correlation coefficient of prediction (rp2) of 0.9072, 0.9922 and 0.9904, respectively, and root mean square error of prediction (RMSEP) of 0.1004, 0.0290 and 0.0323, respectively. Thus, this strategy is very promising in tracking the supply chain of herb-based pharmaceutical industry, especially for identifying adulteration of medicinal materials from their closely related herbal species.

Extract from Eucommia ulmoides Oliv. ameliorates arthritis via regulation of inflammation, synoviocyte proliferation and osteoclastogenesis in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Eucommia ulmoides Oliv., a valuable Chinese herb, has shown a variety of health benefits. Despite the widespread clinical use of this herb to treat rheumatoid arthritis (RA) in Traditional Chinese Medicine (TCM), very few studies have described its anti-pathological effects or mechanism in RA. The present study investigated the mechanism of Eucommia ulmoides Oliv. in an experimental collagen-induced arthritis (CIA) rat model. MATERIALS AND METHODS: The effects of four different Eucommia ulmoides Oliv. extracts on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were screened in an MTT assay, and apoptotic effects were detected by flow cytometry. Among the extracts, the 70% ethanol extract (EU70) presented the best inhibition and was further investigated for its curative effect in CIA rats. Foot swelling was detected, and the arthritis index (AI) was scored. Pathological improvement was assessed by haematoxylin and eosin (H&E) staining of joint tissues. The mechanistic effects of EU70 were investigated as follows: anti-inflammatory effects in Th17-positive cells by flow cytometry; serum levels of inflammatory cytokines by ELISA; TNFα and IL-1ß expression by immunohistochemistry (IHC); and anti-osteoclastogenesis by QPCR detection of RANKL and OPG mRNA. RESULTS: Compared with vehicle treatment in CIA model rats, EU70 significantly ameliorated foot swelling, decreased AI in vivo and reduced inflammatory cell infiltration and synoviocyte proliferation. EU70 decreased the number of Th17-positive cells in the spleen and the serum levels of cytokines, including IL-17, IL-1ß and TNFα, and upregulated the serum levels of IL-10; these results indicated the anti-inflammatory effect of EU70. Moreover, EU70 effectively suppressed TNFα and IL-1ß expression in the joint tissues and resulted in the downregulation of RANKL mRNA and the upregulation of OPG mRNA. These results revealed the possible preventive role of EU70 against bone destruction. CONCLUSION: For the first time, these mechanisms and pathological improvements support the clinical use of Eucommia ulmoides Oliv. in treating RA. The findings indicated that the 70% ethanol extract of Eucommia ulmoides Oliv. could relieve RA symptoms by (1) suppressing the proliferation of synoviocytes, (2) reducing the number of Th17-positive cells and downregulating serum IL-17 expression, (3) increasing the anti-inflammatory effects of IL-10, (4) inhibiting the serum and tissue levels of key pro-inflammatory cytokines, TNFα and IL-1ß, and (5) reducing the degradation of cartilage and bone.

Posttranscriptional regulation of gene expression in epithelial cells by polyamines.

In addition to regulating gene transcription, polyamines also potently modulate gene expression posttranscriptionally. Posttranscriptional gene regulation, which includes processes such as mRNA transport, turnover, and translation, involves specific mRNA sequences (cis-element) that interact with transacting factors such as RNA-binding proteins (RBPs) and microRNAs. U- or AU-rich elements (ARE) are the best characterized cis-acting sequences located in the 3'-untranslated regions of many labile mRNAs. Several RBPs, including AUF1, BRF1, TTP, and KSRP, promote ARE-mRNA decay through the recruitment of the ARE-bearing mRNA to sites of mRNA degradation, whereas RBPs such as HuR, HuB, HuC, and HuD stabilize target mRNAs and stimulate their translation. HuR is one of the best-studied RBPs and has emerged as a key regulator of posttranscriptional control of gene expression and its activity is tightly regulated by cellular polyamines. Ribonucleoprotein immunoprecipitation assays and biotin pull-down assays are two major methods used extensively in experiments investigating the roles and mechanisms of cellular polyamines in the posttranscriptional regulation and are described in detail in this chapter.