O-benzene Sulfonate Research Articles

Inhibition of GRK2 ameliorates the pristane-induced mouse SLE model by suppressing plasma cells differentiation

Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by diverse clinical manifestations and organ damage. Despite its elusive etiology, dysregulated subsets and functions of B cells are pivotal in SLE pathogenesis. Peoniflorin-6′-O-benzene sulfonate (CP-25), an esterification modification of Paeoniflorin, exhibits potent anti-inflammatory and immunomodulatory properties in autoimmune diseases (AID). However, the involvement of CP-25 and its target, GRK2, in SLE development has not been explored. In this study, we demonstrate that both genetic deficiency and pharmacological inhibition of GRK2 attenuate autoantibodies production, reduce systemic inflammation, and mitigate histopathological alterations in the spleen and kidney in the pristane-induced mouse SLE model. Importantly, our findings highlight that both genetic deficiency and pharmacological inhibition of GRK2 suppress plasma cells generation and restore dysregulated B-cell subsets by modulating two crucial transcription factors, Blimp1 and IRF4. Collectively, targeting GRK2 with CP-25 emerges as a promising therapeutic approach for SLE.

CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the β2-adrenergic receptor

In essence, the β2 adrenergic receptor (β2AR) plays an antiproliferative role by increasing the intracellular cyclic 3’,5’-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, β2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the β2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired β2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to β2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not β-arrestin2 or protein kinase A-mediated phosphorylation of β2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6′-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to β2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in β2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA.7HWufKwpH5ZfY_hx8fyY6aVideo

CP-25 exerts a protective effect against ConA-induced hepatitis via regulating inflammation and immune response.

Hepatitis is a complex multifactorial pathological disorder, which can eventually lead to liver failure and even potentially be life threatening. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has proven to have critical anti-inflammatory effects in arthritis. However, the effects of CP-25 in the pathogenesis of hepatitis remains unclear. In this experiment, mice were intragastrically administered with CP-25 (25, 50 and 100mg/kg), and then ConA (25mg/kg) was intravenous injected to establish hepatitis model in vivo. CP-25 administration attenuated liver damage and decreased ALT and AST activities in mice with hepatitis. Besides, CP-25 modulated immune responses including down-regulated the proportions of activated CD4+, activated CD8+ T cells, and ratio of Th1/Th2 in ConA-injected mice. Furthermore, ConA-mediated production of reactive oxygen species (ROS), release of inflammatory cytokines including IFN-γ, TNF-α, activation of MAPK pathways and nuclear translocation of nuclear factor-kappaB (NF-κB) were significantly decreased in CP-25 administrated mice. In ConA-stimulated RAW264.7 cells, CP-25 suppressed inflammatory cytokines secretion and reduced ROS level, which were consistent with animal experiments. Otherwise, the data showed that CP-25 restrained phosphorylation of ERK, JNK and p38 MAPK pathways influenced by ROS, accompanied with inhibiting NF-κB nuclear translocation. In conclusion, our findings indicated that CP-25 protected against ConA-induced hepatitis may through modulating immune responses and attenuating ROS-mediated inflammation via the MAPK/NF-κB signaling pathway.

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6′-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-β-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Paeoniflorin-6'-O-benzene sulfonate protected the intestinal epithelial barrier by restoring the inhibitory effect of GRK2 and β-arrestin 2 on ERK1/2-NF-κB.

Peoniflorin-6'-O-benzene sulfonate (CP-25) inhibited the activity of GRK2 to exert anti-inflammatory and immunomodulatory effects. This study aimed to investigate the effect of CP-25 the intestinal epithelial barrier and the mechanism. CaCO-2 cell monolayer and dextran sulfate salt (DSS)-induced colitis mouse model was used to evaluate intestinal epithelial barrier function in vitro and in vivo, respectively. Results showed that CP-25 prevented dysfunction of the intestinal epithelial barrier and inhibited NF-κB p65 activation in TNF-α-induced CaCO-2 cells. The colon structure destroyed in DSS-induced colitis mice was improved by CP-25. CP-25 has a role in inhibition membrane translocation of GRK2-β-arrestin 2 complex, stabilization of the binding of GRK2 and β-arrestin 2 to ERK1/2 in cytoplasm. Subsequently down-regulated the nuclear transcription and transactivation of NF-κB p65 via inhibiting its phosphorylation of Ser536, and Ser276, respectively and restored the epithelial barrier function. In conclusion, CP-25 inhibited ERK1/2-NF-κB activation and thereby protected the intestinal epithelial barrier, which was associated with restoring the inhibition of GRK2 and β-arrestin 2 on ERK1/2.

CP-25 enhances OAT1-mediated absorption of methotrexate in synoviocytes of collagen-induced arthritis rats.

Organic anion transporter 1 (OAT1) plays a major role in mediating the absorption, distribution and excretion of drugs and other xenobiotics in the human body. In this study we explored the OAT1 status in rheumatoid arthritis (RA) patients and arthritic animals and its role in regulating the anti-arthritic activity of methotrexate (MTX). We showed that OAT1 expression was significantly downregulated in synovial tissues from RA patients compared with that in the control patients. In collagen-induced arthritis (CIA) rats, synovial OAT1 expression was significantly decreased compared with the control rats. In synoviocytes isolated from CIA rats, PGE2 (0.003-1.75 μM) dose-dependently downregulated OAT1 expression, resulting in decreased absorption of MTX. Silencing OAT1 in synoviocytes caused a 43.7% reduction in the uptake of MTX. Furthermore, knockdown of OAT1 impaired MTX-induced inhibitory effects on the viability and migration of synoviocytes isolated from CIA rats. Moreover, injection of OAT1-shRNA into articular cavity of CIA rats significantly decreased synovial OAT1 expression and impaired the anti-arthritic action of MTX, while injection of lentivirus containing OAT1 sequences led to the opposite results. Interestingly, we found that paeoniflorin-6'-O-benzene sulfonate (CP-25) upregulated OAT1 expression both in vitro and in vivo and promoted MTX uptake by synoviocytes via regulating OAT1 expression and function. Taken together, OAT1 plays a major role in regulating MTX uptake by synoviocytes and the anti-arthritic activity of MTX. OAT1 is downregulated in RA and CIA rats, which can be improved by CP-25.

Paeoniflorin-6'O-benzene sulfonate suppresses fibroblast-like synoviocytes proliferation and migration in rheumatoid arthritis through regulating GRK2-Gβγ interaction.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Enhanced G protein coupled receptor kinase 2 (GRK2) translocation and prostaglandin E4 receptor (EP4) desensitization play a critical role in fibroblast-like synoviocytes (FLS) dysfunction. Paeoniflorin-6'O-benzene sulfonate (CP-25) exerts a protective effect in arthritis in the RA animal models. To demonstrate the role of Gβγ in EP4 desensitization and the mechanisms of CP-25 that protects FLS in RA, RA-FLS and adjuvant-induced arthritis (AA-FLS) were isolated from synovium of RA patients and AA rats. RA-FLS, AA-FLS and MH7A were treated with CP-25, Gβγ agonist and antagonist. The cell membrane expression of EP4, GRK2, and Gβγ were detected using western blot analysis. Co-immunoprecipitation (Co-IP) and immunofluorescence were adopted to detect the interactions of GRK2-Gβγ, GRK2-EP4, and EP4-Gβγ. Cell Counting Kit-8 and Transwell assay were used to analyze the proliferation and migration of the FLS. An increased membrane expression of GRK2 and Gβγ, enhanced GRK2-Gβγ interaction and decreased EP4 membrane expression in the RA synovial tissue were identified. In vitro, prostaglandin E2 (PGE2) enhanced the proliferation and migration of FLS. CP-25 exhibited an inhibition effect similar to Gβγ inhibitor, which downregulated GRK2-EP4 interaction, blocked the translocation of GRK2, and reversed EP4 desensitization, leading to the suppression of the proliferation and migration induced by PGE2. These results elucidated that an enhanced GRK2-Gβγ interaction was involved in the EP4 desensitization and dysfunction. CP-25 regulated EP4-GRK2-Gβγ signaling and re-sensitized EP4 by inhibiting GRK2-Gβγ interaction. The regulation of EP4-Gβγ-GRK2 signaling may be a novel potential therapeutic target in RA.

Paeoniflorin-6′-o-benzene sulfonate ameliorates the progression of adjuvant-induced arthritis by inhibiting the interaction between Ahr and GRK2 of fibroblast-like synoviocytes

Aryl hydrocarbon receptor (Ahr) is thought to be a crucial factor that regulates immune responses, which may be involved in the pathogenesis of autoimmune inflammation including rheumatoid arthritis (RA). The results of our group in recent years have shown that Paeoniflorin-6′-O-benzene sulfonate (code: CP-25), a novel ester derivative of paeoniflorin, has a good effect on improving RA animal models. However, whether the anti-arthritis effect of CP-25 is related to Ahr remains unclear. Here, we showed that CP-25 treatment ameliorated adjuvant-induced arthritis (AA), a rat model of RA, by inhibiting Ahr-related activities in fibroblasts like synoviocytes (FLS). AA rats were treated with CP-25 or paroxetine from days 17 to 33 after immunization. We showed that CP-25 alleviated arthritis symptoms and the pathological changes. Treatment with CP-25 decreased the expression of Ahr in the synovium of AA rats. CP-25 inhibited the expression of Ahr and the G protein-coupled receptor kinase 2 (GRK2) as well as the co-expression of GRK2 with Ahr in FLS of AA rats. Furthermore, CP-25 down-regulated the production of Kyn in FLS of AA rats. These results suggested that CP-25 may inhibit the expression and activation of Ahr. Besides, treatment with CP-25 reduced the proliferation and migration of MH7A caused by Ahr activation. In addition, we also demonstrated that CP-25 down-regulated the total and nuclear expression of Ahr and the expression of GRK2 in Kyn-treated MH7A. Moreover, the co-expression and co-localization of Ahr and GRK2in Kyn-treated MH7A were also repressed by CP-25. The data presented here demonstrated that CP-25 suppressed FLS dysfunction in rats with AA, which were associated with reduced Ahr activation and the interaction between Ahr and GRK2.

Synergistic effects of CP-25 and 5-fluorouracil on the hepatocellular carcinoma in vivo and in vitro through regulating activating mitochondrial pathway.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) has therapeutic potential for the treatment of hepatocellular carcinoma (HCC). 5-Fluorouracil (5-Fu) has been a conventional chemotherapeutic agent for HCC. Unfortunately, the nonspecific cytotoxicity and multidrug resistance caused by long-term use limited the clinical efficacy of 5-Fu. This study was aimed to investigate whether the combination of CP-25 and 5-Fu could generate synergistic effect in inhibiting HCC. The experiments on the diethylnitrosamine (DEN) -induced mice showed that compared with applying single drugs, the combination of CP-25 and 5-Fu presented stronger inhibition in tumor nodule and volume. Meanwhile, CP-25 and 5-Fu activated the intrinsic mitochondrial apoptosis pathway induced by P53, inhibited anti-apoptotic B-cell lymphoma (Bcl-2), induced the pro-apoptotic Bcl-2-associated X protein (Bax), Cytochrome-C and caspases. In addition, the synergistic effect was also validated in Bel-7402 and HepG-2 cells in vitro. This research not only provides a novel and effective combination strategy for the therapy of HCC but also provides an experimental basis for the development of CP-25 and 5-Fu compound preparation.

Effects of paeoniflorin-6'-O-benzene sulfonate on the pharmacokinetics, excretion and tissue distribution of leflunomide in rats.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a novel ester derivative of paeoniflorin, which has been shown to have synergistic pharmacodynamic effects with leflunomide (LEF). To determine the effects of CP-25 on the pharmacokinetics of LEF in rats, we developed a ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based method for the determination of levels of teriflunomide (TER, an active metabolite of LEF). This method was used to determine TER concentrations in the plasma, urine, faeces and bile; heart, liver, spleen, lung, kidney, intestinal, brain and synovial tissues; and peripheral blood mononuclear cells (PBMCs) of rats in the control (LEF [10mg/kg]) and combined (CP-25 [50 mg/kg × 7d] plus LEF [10mg/kg]) groups. TER area under the curve [AUC], Tmax , mean residence time (MRT), t1/2α and t1/2β were significantly lower, and clearance (CL) was significantly higher in the combined group than in the control group. Oral CP-25 administration in combination with LEF was found to promote TER excretion in urine, faeces and bile and to reduce its contents in most tissues and organs, especially in the liver, which may reduce LEF-induced liver injury. CP-25 also increased TER exposure in the synovium and its absorption by PBMCs, and this could explain the synergistic effects of CP-25 and LEF.

CP-25 exerts therapeutic effects in mice with dextran sodium sulfate-induced colitis by inhibiting GRK2 translocation to downregulate the TLR4-NF-κB-NLRP3 inflammasome signaling pathway in macrophages.

Deficiency of G protein-coupled receptor kinase 2 (GRK2) was found to protect mice from dextran sulfate sodium (DSS)-induced colitis. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has been shown to exert anti-inflammatory immune regulatory effects in animal models of inflammatory autoimmune disease. This study aimed to investigate the of GRK2 in the pathogenesis of ulcerative colitis (UC) and its effects on macrophage polarization, macrophage subtype regulation of intestinal barrier function, and therapeutic effects of CP-25 in mice with DSS-induced colitis. We found imbalanced macrophage polarization, intestinal barrier dysfunction, and abnormal activation of GRK2 and TLR4-NF-κB-NLRP3 inflammasome signaling pathway in the colonic mucosa of patients with UC. CP-25, restored the damaged intestinal barrier function by inhibiting the transmembrane region of GRK2 in macrophages stimulated by lipopolysaccharides. CP-25 exerted therapeutic effects by ameliorating clinical manifestation, regulating macrophage polarization, and restoring abnormally activated TLR4-NF-κB-NLRP3 inflammasome signaling pathway by inhibiting GRK2. These data suggest the pathogenesis of UC may be related to the imbalance of macrophage polarization, which leads to abnormal activation of TLR4-NF-κB-NLRP3 inflammasome signaling pathway mediated by GRK2 and destruction of the intestinal mucosal barrier. CP-25 confers therapeutic effects on colitis by inhibiting GRK2 translocation to induce the downregulation of TLR4-NF-κB-NLRP3 inflammasome signaling in macrophages.

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6′-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.The therapeutic effects of CP-25 on experimental Sjögren's syndrome has been shown to be associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. The study provides an experimental foundation for CP-25 as a potential drug in the treatment of human autoimmune disorder, Sjögren's syndrome.

CP-25 ameliorates methotrexate induced nephrotoxicity via improving renal apoptosis and methotrexate excretion

Paeoniflorin-6′-O-benzene sulfonate (CP-25) is a derivative of Paeoniflorin. We investigate beneficial effect of CP-25 on methotrexate (MTX) induced nephrotoxicity in rats. Plasma blood urea nitrogen (Bun), plasma creatinine (CREA), urine CREA and protein in the rats were quantitatively measured. Renal tissues were pathologically observed, and apoptosis was detected. Apoptosis related proteins and organic anion transporter-3 (OAT3) expression were determined by western blotting analysis. MTX induced nephrotoxicity and hematotoxicity in rats with abnormal levels of serum Bun, serum CERA, 24 h urine protein excretion, white blood cells, platelets, plateletcrit and abnormal renal pathological appearance. Either pre-treatment or treatment of CP-25 restored normal levels of hematological and renal function parameters, and improved histopathology in rats treated with MTX. CP-25 prevented MTX induced apoptosis of renal tubular cells, and the effect was further confirmed by its regulatory effects on abnormal expression of Bax, cleaved-caspase-3, cleaved-caspase-8, Cyt-c, Bcl-2. The other important finding is co-administration of CP-25 with MTX significantly increased MTX renal excretion in the damaged rats, and the effect is supposed to be linked with its regulation on abnormal renal OAT3 expression. Taken together, CP-25 shows well protective activity against MTX induced nephrotoxicity, and this effect is via its anti-apoptosis and detoxification properties.

Paeoniflorin-6'-o-benzene sulfonate (CP-25) improves vasculitis through inhibiting IL-17A/JAK/STAT3 signaling pathway in endothelial cells of HFD CIA rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects not only joints but also multiple organ systems including cardiovascular system. Endothelial dysfunction plays an important role in cardiovascular diseases (CVD). In RA, endothelial dysfunction exists at both the macrovascular and the microvascular levels, which is a precursor to vasculitis. This study aimed to investigate the pathogenesis of vasculitis and the therapeutic effect of CP-25 on vasculitis in high-fat diet (HFD) collagen-induced arthritis (CIA) rats. Experimental groups were divided into normal group, HFD group, CIA group, HFD CIA group, CP-25 group and MTX group. In vitro, IL-17A was used to stimulate human umbilical vein endothelial cells (HUVECs), and then CP-25 was used to intervene. Results showed that CP-25 reduced global scoring (GS), arthritis index (AI), and swollen joint count (SJC) scores, improved histopathological score, reduced T cells percentage, and decreased IL-17A and ICAM-1 levels. Besides, CP-25 reduced the expression of p-STAT3 to normal levels in vascular of HFD CIA rats. In vitro, IL-17A promoted the expression of p-JAK1, p-JAK2, p-JAK3, pSTAT3, and ICAM-1, and CP-25 inhibited the expression of p-JAK1, p-JAK2, p-JAK3, p-STAT3, and ICAM-1. In conclusion, CP-25 might inhibit endothelial cell activation through inhibiting IL-17A/JAK/STAT3 signaling pathway, which improves vasculitis in HFD CIA rats.

Inhibitory effect of CP-25 on intimal formation and vascular hyperplasia via suppression of GRK2/ERK1/2/EVI1 signaling

Excessive proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) are the center of intimal formation during in-stent restenosis and vein graft disease. Paeoniflorin-6′-O-benzene sulfonate (CP-25) is known to suppress inflammation and atherogenesis. However, the potential effect of CP-25 on intimal formation remains elusive. In the present study, we found that CP-25 significantly attenuated wire injury-induced intimal formation in C57BL/6 mice (intimal area: 2.64 ± 0.25 × 104 μm2 vs. 1.53 ± 0.21 × 104 μm2, P < 0.05) and vascular hyperplasia indicated by PCNA staining. In vitro experiments showed that CP-25 significantly alleviated human aortic smooth muscle cell (HASMC) proliferation, migration and dedifferentiation induced by PDGF-BB. Mechanistically, CP-25 inhibited GRK2 phosphorylation through PDGF receptor in the presence of PDGF-BB. In accordance with these results, CP-25 disrupted the interaction of GRK2 with ERK1/2 and suppressed the activation of ERK1/2 signaling in HASMCs. EVI1, which is considered as a downstream of ERK1/2 signaling and a novel transcription factor for VSMC differentiation, was also downregulated by CP-25 treatment. Moreover, overexpression of EVI1 partly restored the decreased proliferation and dedifferentiation of HASMCs treated by CP-25. Collectively, these findings suggested that CP-25 could alleviate intimal formation in response to wire injury via suppression of the interaction of GRK2 and ERK1/2 and EVI1 activation, indicating CP-25 might serve as a potent pharmaceutical for intimal formation.

CP-25 improves nephropathy in collagen-induced arthritis rats by inhibiting the renal inflammatory response

Paeoniflorin-6′-O-benzene sulfonate (CP-25) is a derivative of paeoniflorin. We previously confirmed that CP-25 inhibits inflammatory responses in several arthritis animal models. The aim of the present study was to investigate the beneficial effects of CP-25 on renal damage in rats with collagen-induced arthritis (CIA). CIA was induced in rats, which were orally administered CP-25 (25, 50 and 100 mg/kg/day) for 24 days. The levels of plasma blood urea nitrogen (BUN) and urine protein in CIA rats were measured. Pathological changes in renal tissues and joints were observed, and inflammatory cell infiltration was evaluated by immunohistochemistry. Moreover, renal inflammatory mediators and transporters were measured by western blotting. We found that CP-25 not only inhibited arthritis manifestations but also improved renal pathological manifestations and kidney injury by decreasing serum BUN and urine protein levels. Further study revealed that CP-25 treatment reduced the number of renal CD68+ cells and downregulated the levels of MCP-1, TNF-α and IL-6 in CIA rats. On the other hand, we noted that CP-25 decreased the ratios of phosphorylated NF-κB p65 (p-p65) to total p65 and p-IκBα to total IκBα in CIA rats, suggesting that CP-25 blocked NF-κB activation. Finally, we observed that CP-25 restored the abnormal expression of OAT1 and OCT1 in the renal tissues of CIA rat. Our data indicate that CP-25 ameliorates kidney damage in CIA rats, and this beneficial effect is closely related to inhibiting renal inflammation and the abnormal expression of transporters.

CP-25, a compound derived from paeoniflorin: research advance on its pharmacological actions and mechanisms in the treatment of inflammation and immune diseases.

Total glycoside of paeony (TGP) has been widely used to treat inflammation and immune diseases in China. Paeoniflorin (Pae) is the major active component of TGP. Although TGP has few adverse drug reactions, the slow onset and low bioavailability of Pae limit its clinical use. Enhanced efficacy without increased toxicity is pursued in developing new agents for inflammation and immune diseases. As a result, paeoniflorin-6'-O-benzene sulfonate (CP-25) derived from Pae, is developed in our group, and exhibits superior bioavailability and efficacy than Pae. Here we describe the development process and research advance on CP-25. The pharmacokinetic parameters of CP-25 and Pae were compared in vivo and in vitro. CP-25 was also compared with the first-line drugs methotrexate, leflunomide, and hydroxychloroquine in their efficacy and adverse effects in arthritis animal models and experimental Sjögren's syndrome. We summarize the regulatory effects of CP-25 on inflammation and immune-related cells, elucidate the possible mechanisms, and analyze the therapeutic prospects of CP-25 ininflammation and immune diseases, as well as the diseases related to its potential target G-protein-coupled receptor kinases 2 (GRK2).This review suggests that CP-25 is a promising agent in the treatment of inflammation and immune diseases, which requires extensive investigation in the future. Meanwhile, this review provides new ideas about the development of anti-inflammatory immune drugs.

Paeoniflorin-6′-O-benzene sulfonate down-regulates CXCR4-Gβγ-PI3K/AKT mediated migration in fibroblast-like synoviocytes of rheumatoid arthritis by inhibiting GRK2 translocation

ObjectiveThis study aims to explore the effect of paeoniflorin-6′-O-benzene sulfonate (CP-25) on the migration of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and the mechanism focused on CXCR4-Gβγ-PI3K/AKT signaling. MethodsHuman synovial tissues were collected from RA and osteoarthritis (OA) patients. Immunohistochemistry (IHC) and Western blot were used to detect the protein expression of CXCR4, GRK2, Gβγ, PI3K, p-PI3K, AKT and p-AKT. Transwell was adopted to analyse the migration of fibroblast-like synoviocytes (FLS). Co-immunoprecipitation (Co-IP) and laser scanning confocal microscopy (LSCM) were used to detect the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ. ResultsThe expression level of CXCR4, GRK2, Gβγ, p-p85 and p-AKT were increased in RA synovial tissue according to the results of IHC and Western blot. In vitro, the migration of FLS was increased after stimulation of CXCL12, inhibition of Gβγ suppressed the migration and phosphorylation of p85 and AKT induced by CXCL12 in FLS, and CP-25 had the same effect as inhibition of Gβγ. The membrane expression of GRK2, Gβγ, PI3K and the combination of GRK2 and Gβγ, the combination of PI3K and Gβγ in FLS were increased after the stimulation of CXCL12, and CP-25 had an ability in reducing the membrane expression and the combination of these proteins. ConclusionExcessive migration of FLS in RA was associated with over-activation of PI3K/AKT signaling, and the activity of Gβγ was involved in the over-activation of PI3K/AKT. CP-25 down-regulated CXCR4-Gβγ-PI3K/AKT signals by inhibiting GRK2-Gβγ complex membrane translocation.

CP-25 inhibits PGE2-induced angiogenesis by down-regulating EP4/AC/cAMP/PKA-mediated GRK2 translocation.

G protein-coupled receptor kinase 2 (GRK2), a type of cytosolic enzyme, transiently translocates to the plasma membrane upon G protein-coupled receptors (GPCRs) activation, and it also binds to extracellular signal-regulated kinase (ERK) to inhibit the activation of ERK. GRK2 deficiency in endothelial cells (ECs) leads to increased pro-inflammatory signaling and promotes recruitment of leukocytes to activated ECs. However, the role of GRK2 in regulating angiogenesis remains unclear. Here, we show that GRK2 is a novel regulatory molecule on migration and tube formation of ECs, vessel sprouting ex vivo and angiogenesis in vivo. We identify that EP4/AC/cAMP/protein kinase A (PKA)-mediated GRK2 translocation to cells membrane decreases the binding of GRK2 and ERK1/2 to inhibit ERK1/2 activation, which promotes prostaglandin E2 (PGE2)-induced angiogenesis. GRK2 small interfering RNA (siRNA) inhibits the increase in PGE2-induced HUVECs migration and tube formation. In vivo, PGE2 increases ECs sprouting from normal murine aortic segments and angiogenesis in mice, but not from GRK2-deficient ones, on Matrigel. Further research found that Lys220 and Ser685 of GRK2 play an important role in angiogenesis by regulating GRK2 translocation. Paeoniflorin-6'-O-benzene sulfonate (CP-25), as a novel ester derivative of paeoniflorin (pae), has therapeutic potential for the treatment of adjuvant arthritis (AA) and collagen-induced arthritis (CIA), but the underlying mechanism of CP-25 on angiogenesis has not been elucidated. In our study, CP-25 inhibits the migration and tube formation of HUVECs, and angiogenesis in mice by down-regulating GRK2 translocation activation without affecting GRK2 total expression. Taken together, the present results revealed that CP-25 down-regulates EP4/AC/cAMP/PKA-mediated GRK2 translocation, restoring the inhibition of GRK2 for ERK1/2, thereby inhibiting PGE2-stimulated angiogenesis.

Alleviating effect of paeoniflorin-6′-O-benzene sulfonate in antigen-induced experimental Sjögren’s syndrome by modulating B lymphocyte migration via CXCR5-GRK2-ERK/p38 signaling pathway

Primary Sjögren's syndrome (pSS) is an autoimmune disease of unresolved aetiology that affects the exocrine glands. Clinical symptoms frequently also involve skin, liver, kidney and neurovascular components. The pathogenesis of pSS is still unclear but B cell hyperactivity has been identified as a hallmark of pSS. Currently, a curative therapeutic agent is lacking. In this study, we explored whether paeoniflorin-6′-O-benzene (CP-25) exerted therapeutic effects through regulating B lymphocyte migration via CXCR5-GRK2-MAPK mediated signaling pathways in a mouse model of antigen-induced, experimental Sjögren's syndrome (ESS). We found that CP-25 increased the salivary flow and alleviated the histopathology of ESS. Furthermore, CP-25 reduced the viability of B lymphocyte and limited the target organs index. In the peripheral blood and salivary gland of ESS mice, CP-25 down-regulated the proportion of total B cells, CXCR5+ B cells and PDCA1 + CD19- and limited the presence of phosphorylated (p-) p38 and ERK (p-ERK). Besides, CP-25 increased the percentage of memory B cells in the peripheral blood and reduced it in salivary gland. Furthermore, in vitro, CP-25 down-regulated p-p38, p-ERK, CXCR5 and membrane GRK2, and increased cytoplasm GRK2 in Maver-1 cells, a mantle cell lymphoma cell line, causing a lower migration ability of Maver-1 cells. Thus, we define CP-25 as a novel compound that is a potent therapeutic agent for pSS which modulates B lymphocyte subsets and impacts the migration of B lymphocytes through regulating the CXCR5-GRK2-ERK/p38 signaling pathway.