Targeting the alternative bile acid synthetic pathway for metabolic diseases

The gut microbiota is profoundly involved in glucose and lipid metabolism, in part by regulating bile acid (BA) metabolism and affecting multiple BA-receptor signaling pathways. BAs are synthesized in the liver by multi-step reactions catalyzed via two distinct routes, the classical pathway (producing the 12α-hydroxylated primary BA, cholic acid), and the alternative pathway (producing the non-12α-hydroxylated primary BA, chenodeoxycholic acid). BA synthesis and excretion is a major pathway of cholesterol and lipid catabolism, and thus, is implicated in a variety of metabolic diseases including obesity, insulin resistance, and nonalcoholic fatty liver disease. Additionally, both oxysterols and BAs function as signaling molecules that activate multiple nuclear and membrane receptor-mediated signaling pathways in various tissues, regulating glucose, lipid homeostasis, inflammation, and energy expenditure. Modulating BA synthesis and composition to regulate BA signaling is an interesting and novel direction for developing therapies for metabolic disease. In this review, we summarize the most recent findings on the role of BA synthetic pathways, with a focus on the role of the alternative pathway, which has been under-investigated, in treating hyperglycemia and fatty liver disease. We also discuss future perspectives to develop promising pharmacological strategies targeting the alternative BA synthetic pathway for the treatment of metabolic diseases.

Intervention effect of tetramethylpyrazine on inflammatory response of endothelial cells induced by activated complement alternative pathway / 中国药理学通报

Aim To study the intervention effect of tet- ramethylpyrazine(TMP) on human microvascular endothelial cells (HMECs) inflammatory response induced by activated complement alternative pathway and the possible molecular mechanisms. Methods HMECs were pretreated with different concentrations of TMP, and then exposed to the activated products of the complement alternative pathway which was prepared by cobra venom factor( CVF). The supernatant was removed and assayed for expression of the adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and the inflammatory mediator(IL-6 and TNF-ot) by using ELISA reagent kits. The nucleus transcriptional activity of NF- kB was measured by the dual luciferase reporter assay ? system. Results The adhesion molecules, inflammato ry mediator and nucleus transcriptional activity of NF- kB increased after HMECs were exposed to the products of the activated complement alternative pathway. The up-regulation of ICAM-1, VCAM-1, E-selectin, IL-6, TNF-a and the nucleus transcriptional activity of NF-kB were inhibited by various concentrations of TMP in a dose-dependent manner. Conclusions TMP can effectively reduce inflammatory response of HMECs in-duced by the activated complement alternative pathway , and the mechanism may be highly related to inhibition of nucleus transcriptional activity of NF-kB.

Effects of icariin on acute lung injury in mice induced by activation of the complement alternative pathway / 中国药理学通报

Aim To explore the protective effect and mechanism of icariin ( ICA ) on acute lung injury ( ALI) in mice induced by activation of the comple-ment alternative pathway. Methods 32 healthy KM mice were randomly divided into four groups: the nor-mal group, the model group, the PDTC group and the Icariin group, which received 7-day intragastric admin-istration respectively. Then cobra venom factor ( CVF) was used to activate specifically complement alternative pathway to induce acute lung injury in mice by intrave-nous injection. Myeloperoxidase ( MPO ) activity of lung homogenate, the cell count and the protein con- tent of bronchoalveolar lavage fluid ( BALF ) were measured. The concentration of IL-6, TNF-α, P-selec-tin and ICAM-1 in BALF and serum were determined by ELISA. The pathological change of lung tissue was observed by HE staining. The phosphorylation of NF-κB p65 in lung tissues was checked by immunohisto-chemistry. The effect of the transcriptional activity of NF-κB signal pathway in microvascular endothelial cells was measured by employing dual-luciferase re-porter assay system. Results ICA reduced MPO ac-tivity of lung homogenate, the cell count and the con-tent of IL-6, TNF-α, P-selectin in BALF obviously. The level of TNF-α, P-selectin and ICAM-1 in serum was decreased, the pulmonary inflammatory cell infil-tration was reduced, the phosphorylation of NF-κB p65 in lung was inhibited significantly and the transcrip-tional activity of NF-κB was also down-regulated. Con-clusion ICA can alleviate acute inflammatory re-sponse of ALI mice induced by activation of the com-plement alternative pathway. The mechanism may be highly related to the inhibition of inflammatory cell in-filtration in lung tissue, the down-regulation of phos-phorylation of NF-κB p65 and nuclear transcriptional activity.

Advances in research of mannan-binding lectin-associated serine protease 3 / 中国免疫学杂志

MASP3 is one of the mannan-binding lectin-associated serine proteases(MASPs)of the complement lectin pathway.It is the alternative splice product of MASP1/3 gene.MASP3 does not participate in the activation of the lectin pathway and it has no cleavage activity towards C2,C4 and C3.Recently,a novel MASP3 function has been discovered that it may play an important role in the alternative pathway through direct activation of factor D.Besides,studies on the murine disease model suggested that MASP3 might be a new therapeutic target for the treatment of alternative pathway-mediated diseases.This review present an overall description of the coding gene and protein structure of MASP3 protein and provide recent research progress on its function and especially in its role in the alternative pathway-mediated diseases.

Complement in ANCA-associated vasculitis / 中华检验医学杂志

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases with rapid progession and poor prognosis.The histopathological hallmark in the kidneys of patients with AAV is " pauci-immune" necrotizing crescentic glomerulonephritis,therefore complement was previously suggested not to play a major role in the disease development.However,in the recent years,accumulating evidence from both experimental and clinic study has strongly incriminated alternative complement pathway activation as critically important in the pathogenesis of AAV.In patients with AAV,plasma levels of Bb and FH,components of the alternative complement pathway,are associated with disease activity and prognosis,which might be useful biomarkers in monitoring systemic disease activity and renal disease activity in AAV.The complement activation product C5a and its interaction with C5a receptor play a central role.It is suggested that neutrophils,ANCA and complement system form a positive feedback loop contributing to the development of AAV.Preliminary data of two clinical trials have demonstrated effectiveness and safety of C5a receptor blockade in patients with AAV.Therefore,measurement of complement biomarkers will be helpful in assessing the disease activity of patients and be of great importance for monitoring the efficiency of complement-targeting therapy in the future.

Effect of chlorogenic acid,caffeic acid,and ferulic acid on inhibition of inflammatory response of HMECs induced by activated complement alternative pathway / 中国药理学通报

Aim To investigate the effect of chlorogen-ic acid,caffeic acid,and ferulic acid on expression of molecules related with inflammatory response of HMECs induced by activated complement alternative pathway.Methods CVF was used to activate the al-ternative pathway of serum complement.After exposure of HMECs to activate complement for various times, supernatant of cell culture was removed and assayed for content of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 u-sing ELISA kits.The expression of the above mole-cules induced by activated complement was measured after HMECs were pre-treated with 50,100,250 μmol ·L-1 of CGA,CA,and FA.Results After HMECs were exposed to the product of the activated comple-ment alternative pathway,the expression of ICAM-1 , IL-6,IL-8,t-PA,and PAI-1 was up-regulated.The expression of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 was down-regulated by various concentrations of CGA, CA,and FA.ICAM-1 and IL-8 were inhibited most significantly in all molecules mentioned above.CA ex-hibited the best intervention effect,followed by FA. Conclusion Certain concentration of CGA,CA,and FA can inhibit the expression of ICAM-1,IL-6,IL-8, t-PA,and PAI-1 in HMECs induced by the activation of the alternative complement pathway,indicating that CGA,CA,and FA can inhibit inflammatory response of HMECs.

Effect of three chemical molecules on adhesion molecules expression in HMECs induced by activated complement alternative pathway / 中国药理学通报

Aim To investigate the effect of resveratrol ( Res) , PDTC and AG490 on adhesion molecules ex-pression induced by product of activated complement alternative pathway on human microvascular endothelial cells ( HMECs) and the possible mechanisms. Meth-ods HMECs were exposed to the product of comple-ment alternative pathway activation, then the superna-tant was removed to detect the concentration of malond-ialdehyde ( MDA ) with TBA method. ELISA method was used to detect the expression of soluble ICAM-1 , VCAM-1 ( and E-selectin) in the culture supernatant. Res, PDTC and AG490 with different concentrations were used to determine their effect on cell oxidation level and adhesion molecules expression. The phospho-rylation of NF-κB p65 was detected by Western blot, and the intervention of Res, PDTC and AG490 was as-sayed by the same way. Results The activation of complements alternative pathway resulted in the phos-phorylation of NF-κB p65 , and increased the concen-tration of MDA and up-regulated the expression of ICAM-1, VCAM-1 and E-selectin. Res reduced the concentration of MDA. Res, PDTC and AG490 inhibi-ted the phosphorylation of NF-κB p65 . Res and PDTC showed similar inhibition on expression of ICAM-1 and VCAM-1 , while exhibiting little effect on expression of E-selectin, and AG490 significantly inhibited the ex-pression of the above adhesion molecules. Conclusions Res, PDTC and AG490 could inhibit the expression of adhesion molecules induced by activated complement alternative pathway, the inhibition of NF-κB pathway activation was involved in their mechanism, and JAK2 may be a more important intervention target in regula-ting adhesion molecule expression.

Expression of coagulation-and fibrinolysis-related molecules of endothelial cells induced by activated complement alternative pathway and intervention / 中国药理学通报

Aim To investigate the change of molecu-lar expression related to coagulation and fibrinolysis in human microvascular endothelial cells ( HMEC ) in-duced by activated complement alternative pathway and effect of pyrrolidine dithiocarbamate ( PDTC ) and res-veratrol on intervention. Methods Normal human se-rum was activated by cobra venom factor ( CVF) . After exposure of HMEC to activated complement for various times, supernatant was removed and assayed for ex-pressions of P-selectin, VWF, t-PA, PAI-1, TF, TM, and NO by using reagent kits. The expressions of the above molecules in HMEC pretreated with PDTC and resveratrol were also investigated. Results P-selectin and VWF were rapidly released by endothelial cells and the expression reached the peak at the time point of 15 min. The expressions of t-PA, PAI-1, and TF were continuously upregulated, whereas NO and TM were decreased. PDTC and resveratrol inhibited the upregulation of P-selectin, VWF, t-PA, PAI-1 and TF, and intervened the downregulation of NO. Res-veratrol further downregulated the expression of TM. Conclusion Activated complement alternative path-way can influence the expression of molecules related to coagulation and fibrinolysis in HMEC, and PDTC and resveratrol can affect this change.

Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides

Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosorbent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELISA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of suramin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC50 between Bupleurum smithii var. parvifolium polysaccharides (1.055 mg/mL) and Bupleurum chinense polysaccharides (0.98 mg/mL) showed that, similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti-complementary properties against the lectin pathway. The results demonstrate that the described ELISA assay can compensate for the shortcomings of the hemolytic assay in lectin pathway.

Biología comparada del sistema de complemento en peces / Comparative biology of the complement system in fish / Biologia comparativa do sistema complemento em peixes

Dentro de la respuesta inmune humoral se encuentran componentes que mantienen la homeostasis de los organismos a través del control de agentes patógenos por medio de la opsonización, quimiotaxis de células fagocíticas facilitando el proceso de eliminación de lo extraño o sin su acompañamiento, en el caso de la formación de poros en la membrana celular. A un grupo de este conjunto de componentes de origen molecular proteico se denominósistema del complemento, el cual posee tres vías de activación (Clásica, Alternativa y Lectinas), funciona comoanafilatoxinas, reguladores y receptores. La presente revisión tiene como objetivo discutir acerca de los diferentes componentes del sistema del complemento en la escala animal enfocándose principalmente en peces teleósteos y mamíferos, como organismos modelos en busca de elucidar sus diferencias, homologías y respuestas.

Within the humoral immune response can be found components that maintain an organism’s homeostasis viacontrol of pathogenic agents using opsonization, chemotaxis of phagocytic cells which facilitates the processof elimination of foreign bodies, or in its absence, the formation of pores in the cellular membrane. One of these groups of components, of protein origin, is referred to as the complement system, which has 3 means of activation (Classic, Alternative, and Lectins) and functions as anaphylactic toxins, regulators and receptors. The aim of this review is to discuss the different components of the complement system in the animal kingdom, focusing principally on teleost fish and mammals, as model organisms in the search to elucidate their differences, homologies, and answers.

Dentro da resposta imune humoral encontram-se componentes que mantém a homeostase do organismo através docontrole de patógenos, por opsonização, quimiotaxia de células fagocíticas que facilita o processo de eliminaçãode corpos estranhos, ou na sua ausência, a formação de poros na membrana celular. Este conjunto de componentes moleculares de origem protéica são chamados de sistema complemento, que tem três vias de ativação (clássica, alternativa e lectinas), funciona como anafilatoxinas, reguladores e receptores. Esta revisão tem como objetivo discutir os vários componentes do sistema complemento na escala animal focando principalmente em peixes teleósteos e mamíferos como organismos modelos na busca de elucidar suas diferenças, homologias e respostas.

Alternative pathways for angiotensin II generation in the cardiovascular system

The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.