Cyclo(Val-Pro) and Cyclo(Leu-Hydroxy-Pro) from Pseudomonas sp. (ABS-36) alleviates acute and chronic renal injury under in vitro and in vivo models (Ischemic reperfusion and unilateral ureter obstruction).

The study aimed to identify small molecules having potentiality in alleviating renal injury. Two natural compounds cyclo(Val-Pro) (1) and cyclo(Leu-Hydroxy-Pro) (2) were first evaluated under acute renal injury model of ischemic reperfusion at different doses of 25, 50 and 75 mg/kg body weight. Further, the compounds were subjected to antimycin A-induced ischemic in vitro study (NRK-52E cell lines). Both the compounds significantly decreased plasma IL-1ß levels (P < 0.05). Also, the mRNA expression levels of inflammatory markers (TNF-α, IL-6 and IL-1ß) and renal injury markers (KIM-1, NGAL, α-GST and π-GST) in the renal tissues were significantly alleviated (P < 0.01) along with the improvement in histological damage and control over neutrophil infiltration as a result of ischemic reperfusion. The in vitro study revealed the protective effect against antimycin A-induced cytotoxicity (P < 0.05) and antiapoptotic effect acting through the regulation of Bax, caspase 3 (pro and cleaved) and BCL2 with reduction in Annexin+PI+ cells. Further, the compound cyclo(Val-Pro) (1) was evaluated (50 mg/kg body weight dose) in chronic unilateral ureter obstruction model of renal injury in mice and TGF-ß-induced in vitro fibrotic model (NRK-49F cell lines). Cyclo(Val-Pro) (1) significantly reduced the expression levels of fibrotic markers (collagen-1, α-SMA and TGF-ß) and showed marked alleviation of renal fibrosis (sirius red staining). Also, the proliferation of TGF-ß-induced NRK-49F cells was significantly reduced along with decreased levels of collagen-1 and α-SMA in immunohistochemistry studies. In conclusion, the compounds significantly abrogated ischemic injury by inhibiting renal inflammation and tubular epithelial apoptosis. Further, cyclo (Val-Pro) (1) exhibited significant anti-fibrotic activity through the inhibition of fibroblast activation and proliferation. Thus, these proline-based cyclic dipeptides are recommended as drug leads for treating renal injury.

Halodule pinifolia (Seagrass) attenuated lipopolysaccharide-, carrageenan-, and crystal-induced secretion of pro-inflammatory cytokines: mechanism and chemistry.

OBJECTIVES: The study aimed to explore the anti-inflammatory effect, underlying mechanism, and chemistry of Halodule pinifolia extract. METHODS: The ethyl acetate (EHP) and methanol (MHP) extracts of Halodule pinifolia were screened for pro-inflammatory cytokine inhibition effect under various in vitro (LPSand crystal-induced inflammation) and in vivo models (LPS-induced endotoxaemia model, carrageenan-induced paw oedema model, and oxalate-induced renal nephropathy model of inflammation). The effect of EHP on the expression of inflammatory markers using western blot analysis (in vitro) was investigated. Chemical constituents of bioactive EHP were isolated through chromatography and characterised using NMR spectroscopy. Furthermore, EHP was standardised for rosmarinic acid, vanillic acid, and ethyl protocatechuate using HPLC. Also, total phytosterols, phenolic, and flavonoid content of EHP were determined by UV spectroscopy. KEY FINDINGS: EHP was comparatively more effective than MHP in inhibiting cytokines secretions under LPS-induced in vitro models. Furthermore, EHP was screened under endotoxaemia in vivo model, EHP (250 mg/kg) reduced plasma IL-6, TNF-α, and IL-1ß levels by 88.3%, 78.2%, and 74.5%, respectively. In the carrageenan-induced oedema model, EHP (200 mg/kg) reduced paw volume and release of TNF-α (69.3%) and IL-1ß (43.1%). EHP (200 mg/kg) further controlled renal nephropathy by inhibiting plasma IL-1ß and BUN levels. Also, a significant reduction of mRNA expressions of TNF-α and IL-1ß and KIM-1 in renal tissues was observed. Through western blot, EHP was identified to regulate the expression of pro-form as well as mature-form of IL-1ß and caspase-1. EHP constituted rosmarinic acid (RA), vanillic acid (VA), ethyl protocatechuate (EP), sitosterol, stigmasterol, campesterol, and dihydrobrassicasterol. It was determined that 4.6 mg/g of RA, 2.92 mg/g of VA, 0.76 mg/g of EP, 21.7 mg/g of total phenolics, 29.8 mg/g of total flavonoids, and 48.2 mg/g of total phytosterols were present in dry EHP. The presence of anti-inflammatory constituents such as RA, VA, and PE in EHP corroborated the in vitro and in vivo anti-inflammatory activity of EHP. CONCLUSION: The anti-inflammatory property of EHP and its action through attenuation of pan-cytokines suggest that it can be developed into an oral pharmaceutical drug.

Methods for Evaluation of TNF-α Inhibition Effect.

Tumor necrosis factor superfamily (TNFSF) ligands and receptors have distinctive structural characters that link them to cell growth, cell survival, or cell death. Some of these can activate both inflammatory and apoptotic pathways, depending on target cell types and other extrinsic stimuli. Many of the TNF receptor superfamily molecules are expressed in cells of the immune system, which may be central to autoimmune and inflammatory diseases as well as cancer. However, the function of TNFSF members is not just restricted to immune cells. Members of TNFSF have been linked to an array of pathophysiologies, including cancer, neurologic, cardiovascular, pulmonary, autoimmune, and metabolic diseases. TNF-α of TNFSF is a pro-inflammatory cytokine produced by macrophages/monocytes, widely implicated in the pathogenesis of inflammatory disorders. In view of these facts, TNF-α has been recommended as an important target for discovering drugs for autoimmune and inflammatory diseases and cancer. Various cell-based assays to understand the role of TNF-α in inflammation and to estimate the concentrations of TNF-α levels in body fluids such as plasma, synovium, etc., are being followed by researchers. In this chapter, methods of cell viability assay, ELISA assay, RT-PCR, and western blot analysis for estimating LPS-induced TNF-α protein expressions are described in detail.

New synthetic coumarinolignans as attenuators of pro-inflammatory cytokines in LPS-induced sepsis and carrageenan-induced paw oedema models.

OBJECTIVES: The aim of the study was to explore the inhibition efficacy of new synthetic coumarinolignans (SCLs) against the secretion of pro-inflammatory cytokines in two in vivo models of inflammation. METHODS: Four SCLs 1-4 were screened for their pro-inflammatory cytokine inhibitory potential through oral administration at a dose of 50 mg/kg body weight in lipopolysaccharide-induced mouse endotoxaemia and carrageenan-induced mouse paw oedema models. Levels of pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) in blood and paw tissue samples were estimated using ELISA. Paw oedema was measured using a plethysmometer. Results were compared with a natural coumarinolignan, cleomiscosin A (5), and the structure-activity relationship (SAR) was interpreted. RESULTS AND DISCUSSION: Compound 2 had the greatest potential in the endotoxaemia model, exhibiting 66.41%, 62.56% and 43.15% inhibition of plasma IL-1ß, TNFα and IL-6 secretions, respectively. Further dose-dependent study revealed its anti-inflammatory potential even at dose of 10 mg/kg body weight with 24.42% decline in the level of IL-1ß. Nevertheless, SCLs 1, 3 and 4 showed marked inhibitory activity with 57.54%, 51.48% and 62.46% reduction in the levels of IL-1ß, respectively. Moreover, compound 2 decreased the plasma TNFα and IL-1ß levels to 50.03% and 36.58% along with the reduction of paw oedema volume in the local inflammation induced by carrageenan. All compounds including cleomiscosin A (5) were more effective against IL-1ß. By studying SAR, the presence of dihydroxyl groups in the phenyl ring of lignans was identified to be essential for the activity. Also, esterification of lignans and presence of a 4-methyl substituent in the coumarin nucleus were found to play some role in enhancing the activity. CONCLUSION: All four SCLs, especially compound 2, have shown vast potential to emerge out as promising anti-inflammatory drugs.

Structure-based design and synthesis of new 4-methylcoumarin-based lignans as pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) inhibitors.

Suppression of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) along with nitric oxide reduction in RAW 264.7 cells by 7,8-dihydroxy-4-methylcoumarin, ethyl p-coumarate, ethyl caffeate and ethyl ferulate drove us to search structural-analogues of the aforementioned compounds through structure-based drug design. Docking studies revealed that substituted cinnamic acids and their ethyl esters (2-7c) showed higher GoldScore-fitness (GSF) and non-bonding interactions with target proteins than 7,8-dihydroxy-4-methylcoumarin (1a) and 7,8-dihydroxy-5-methylcoumarin (1b). With this background, the methylcoumarins (1a and 1b) and the cinnamic acid derivatives (2-7c) were fused in different permutations and combinations to generate sixty novel fused-cyclic coumarinolignans (FCLs) (8-13k). Docking studies on 8-13k indicated that several FCLs possess higher GSF, interesting active site interactions and distinctive π-π interactions compared to the standards (cleomiscosin A, diclofenac Na and prednisolone). Based on these findings, four novel FCLs (9d, 10d, 11d and 11e) were synthesized and tested for inhibition effect on TNF-α, IL-1ß and IL-6 expressions in LPS and oxalate crystal-induced in-vitro models. Compound 10d exhibited significant effect (P < 0.0001 at 100 µM) with an IC50 value of 8.5 µM against TNF-α. Compound 11e possessed IC50 values of 13.29 µM and 17.94 µM against IL-6 and IL-1ß, respectively. Study on SAR corroborated the requirement of C-4-methyl substituent in the coumarin moiety, dihydroxyl groups in the phenyl ring, and esterification of lignans for potent activity. Additionally, the reported excellent anti-inflammatory activity of cleomiscosin-A-glucoside was corroborated by from the higher GSF and better hydrophobic interactions than cleomsicosin A in the docking study. As an outcome, some novel and potentially active FCLs acting through NFκB and caspase 1 signaling pathways have been discovered as multiple cytokine inhibitors.

l-Proline-based-cyclic dipeptides from Pseudomonas sp. (ABS-36) inhibit pro-inflammatory cytokines and alleviate crystal-induced renal injury in mice.

Study on the constituents of bioactive culture broth extract (CBE) of Pseudomonas sp. (ABS-36) explored the secretion of an array of cyclic dipeptides (CDPs) and twenty of them had been isolated and reported in the present paper. Six major CDPs [(cyclo(Leu-Pro) (1), cyclo(Val-Pro) (2), cyclo(Leu-hydroxy-Pro) (9), cyclo(Pro-Tyr) (10), cyclo(Pro-Ala) (11) and cyclo(Gly-Pro) (12)] exhibited pan cytokine inhibition effect by inhibiting key pro-inflammatory cytokines IL-1ß, TNF-α and IL-6 tested under various cell based assays. With this background, the effect of these six CDPs in treating renal inflammation was screened using crystal-induced nephropathy model in mice at 50 mg/kg body weight through oral administration. cis-Cyclo(Val-Pro) (2) exhibited 57% inhibition of plasma IL-1ß protein expression and 35.2% inhibition of elevated blood urea nitrogen. Further, cis-cyclo(Val-Pro) (2) attenuated renal injury as demonstrated by significant reduction of mRNA expressions of IL-1ß (P < 0.01) and kidney injury marker-1 (P < 0.001). Furthermore, evaluation of tubular-necrosis, -dilation and -cast in the histological sections exhibited moderate protection of renal tissues by cis-cyclo(Val-Pro) (2). All the tested CDPs reduced the nitrite production and were interestingly non-cytotoxic.