Experimental Studies Of The Rosmarinic Acid Treatment Of Mesangial Proliferative Glomerulonephritis

Proliferation of glomerular mesangial cell (GMC) and accumulation of extracellular matrix (ECM) are the common pathohistologial features of mesangioproliferative glomerulonephritis(MsPGN), as well as many types of renal diseases, which ultimately leads to glomerulosclerosis and renal failure. Numerous studies have implicated that cytokines and ploypeptide growth factors, such as transforming growth factor-β₁(TGF-β₁),platelet-derived growth factor-BB(PDGF-BB) and monocyte chemoattractant protein-1 (MCP-1) are the important regulators of GMC proliferation and ECM expansion. All cytokines are produced by inflammatory cells, including macrophages, and are also synthesized and released by the mesangial cells themselves under certain conditions. Several pharmacotherapeutics are used for treating MsPGN, including steroids and immunosuppressive agents. Although these treatments are effective for suppressing inflammatory or GMC proliferation, some occasionally cause inherent and unavoidable side effects. Moreover, these pharmacotherapeutics cannot be used for long periods of time. Hence, traditional herbal medicines may improve treatment and inhibit the progression of chronic glomerular disease, especially in patients with weak disease activity.Rosmarinic acid (RA) is a widely distributed phenolic compound in various Labiatae herbs such as Rosmarinus officinalis (rosemary) and Perilla frutescens (perilla). RA is reported to exhibit pharmacological effects, including antioxidative, antiinflammatory, antithrombotic, antibacterial, antimutagen and immunosuppressive effects. Recent studies have revealed that the inhibitory effects of RA on the proliferation of cultured murine mesangial cells and the suppressive effects of RA on MsPGN or IgA nephropathy model in rats. However, the mechanism of the anti-proliferative effect is unclear, especially with respect to cytokines.In the present study, we evaluated the effects of RA on PDGF-BB induced proliferation in cultured murine mesangial cells and its protective effects in the mesangioproliferative anti-Thy-1.1 nephritis. To determine the possible action mechanism of RA on GMC proliferation, We also examined the expression of TGF-β₁ and MCP-1 in vitro and in vivo. We performed the following two-part study.Partâ… : The effects of rosmarinic acid on rat mesangial cells proliferation and cytokines production induced by PDGF-BB in vitro.Objective: To explore the effects of rosmarinic acid (RA) on the proliferation and transforming growth factor-β₁ (TGF-β₁), monocyte chemoattractant protein-1 (MCP-1) as well as fibronectin (FN) secretion in rat glomerular mesangial cells (GMC).Methods: Rat mesangial cells (HBZY-1) were cultured with recombinant human platelet-derived growth factor-BB (PDGF-BB, 20ng/ml) in presence of different concentration of RA (200,100,50,25,12.5,6.25,3.13,1.57,0.78及0μg/ml), set up the effective and safe concentration of RA use MTT and trypan blue viability test. Then the cultured mesangial cells were divided into five groups: (1)normal group; (2)GMC proliferation group induced by PDGF-BB; (3)three different concentration of RA groups(5μg/ml, 15μg/ml and 25μg/ml, respectively). Collected the supernatant and the cells respectively. The proliferation of GMC was determined by ³H-TdR incorporation. The levels of the MCP-1, TGF-β₁ and FN protein in the supematant were mesured by ELISA, while the MCP-1, TGF-β₁ and FN mRNA expression were detected by semi-quantitative RT-PCR.Results: PDGF-BB could stimulate the proliferation of GMC, upregulate the expression of MCP-1, TGF-β₁ and FN mRNA, and induce the MCP-1, TGF-β₁ as well as FN protein release in rat GMC. Whereas RA could inhibit the mRNA expression or protein production of MCP-1, TGF-β₁ and FN in GMC induced by PDGF-BB in a dose-dependent manner.Conclusion: RA could inhibit the proliferation of GMC, down-regulate the secretion of MCP-1, TGF-β₁ and FN in rat GMC induced by PDGF-BB in vitro. Our results suggest that RA could inhibit the generation of the proinflammatory factors in some renal diseases and might be beneficial in retarding progressive development of glomerulonephritis.Partâ…¡: Protective effects of rosmarinic acid on experimental mesangioproliferative glomerulonephritis by reducing renal TGF-β₁ and MCP-1 expression in vivo.Objective: To explore the effects of rosmarinic acid (RA) on experimental mesangioproliferative glomerulonephritis (MsPGN) and its relation with renal TGF-β₁ and MCP-1 expression.Methods: Rabbit antithymocyte serum (ATS) was produced, and then the MsPGN animal model were prepared in Sprague-Dawley rats by a single intravenous injection of ATS. Twenty-four rats were randomly divided into four groups(n=6, respectively): normal group, treated with rabbit serum and tap water; control group, treated with ATS (10ml/kg ) and tap water; low-dose and high-dose RA groups, treated with ATS (10ml/kg ) and RA ( 25mg·kg^-1·d^-1 and 50mg·kg^-1·d^-1, respectively). RA was administered by gavage from the day of serum injection (day 0) to day 7. Urinary protein was examined. All rats were sacrificed on day 8, when kidneys were collected. The morphologic lesions were observed by PAS stain, and the expression of proliferation cell neutral antigen (PCNA), TGF-β₁ and MCP-1 were detected by immunohistochemistry.Results: (1) Compared to model group, the level of 24-hour proteinuria in low-dose or high-dose RA treated groups decreased significantly. Renal pathologic changes in RA treated groups was also improved. (2) Compared to normal group, glomerular PCNA-positive cells and the accumulation of FN were significantly increased in model group, which were significantly inhibited by low-dose or high-dose of RA. (3) The expression of TGF-β₁ mainly distributed along glomerular vessels and mesangiums. Compared to normal group, the expression of TGF-β₁ was increased significantly in glomeruli of MsPGN rats. After 7 days treatment, RA significantly down-regulated the expression of TGF-β₁ compared to model group. (4) In normal group, the renal tissue almost did not express MCP-1, but it increased significantly in model group, mainly expressed in glomerular mesangiums, especially in crescents. After 7 days treatment, RA could significantly down-regulated the expression of MCP-1 compared to model group.Conclusion: Renoprotective mechanism of RA may be, at least partly, correlated with suppression on overexpression of TGF-β₁ and MCP-1 in renal tissue of MsPGN rats. Hence, we postulated RA might have benefical for preventing and treating glomerulonephritis.