Rosmarinic Acid Inhibits The Renal Tubular Epithelial - Mesenchymal Quality Effects And Mechanisms Of Transdifferentiation,

Renal interstitial fibrosis (RIF) has become common basic channels and major pathology of end-stage renal disease (ESRD). It is more important than glomerulos-clerosis in ESRD. In pathological conditions, tubular epithelial cells can be epithelial-mesenchymal transdifferentiation (EMT). During renal interstitial fibrosis, about 36% myofibroblasts come from tubular epithelial cells, and EMT plays an important role during this progress.Transforming growth factor-β₁ (TGF-β₁) is one of the key cytokines inducing fibrosis, which can independently initiate and complete the entire process of EMT. TGF-β₁ induces EMT through the signal pathway of Smad, PI3K and MAPK (ERK/P38/JNK). Future study found that reactive oxygen species (ROS) took part in TGF-β₁ induced EMT as the upstream signaling molecules of MAPK. In the process of EMT, TGF-β₁ could increase the generation of cellular ROS to phosphorylate Smad2, P38/MAPK, and ERK1/2.Mitochondrial respiration is the major source of ROS, meanwhile, there exits mitochondrial antioxidant defense system to keep the balance of the levels of ROS. Increased generation of ROS or decreased antioxidant defense capacity is a prerequisite for increased net ROS production and oxidative stress. The accumulation of ROS will lead to mitochondrial dysfunction, which will generate even more ROS. Hence, the relationship between mitochondrial and ROS is very close. Protection of mitochondrial function and inhibition of ROS generation may be a new direction to inhibit EMT.Rosmarinic acid (RA) is a widely distributed phenolic compound in various labiatae herbs such as rosmarinus officinalis (rosemary) and perilla frutescens (perilla). RA has a number of important biological functions, including antiviral, antibacterial, anti-inflammatory, antioxidant and so on. Prior studies have revealed that RA has protective effect in MsPGN of rats based on its function of antioxidant; and also it can inhibit the process of renal interstitial fibrosis and EMT.To further demonstrate whether RA could inhibit EMT and its mechanism, we performed the following two-part study in vitro.Partâ… : Effect of rosmarinic acid on transdifferentiation in mouse proximal tubular cells (mPTCs)Objective: To observe the effects of rosmarinic acid (RA) on the tubular epithelial-mesenchymal transition (EMT) by transforming growth factor-Pi (TGF-β₁).Methods: Growth-arrested and synchronized mouse proximal tubular cells (mPTCs) were stimulated with TGF-β₁ in the presence or absence of RA. Cultured cells were divided into 4 groups: A. negative control; B. 25 ug/ml RA; C. 5 ng/ml TGF-β₁; D. 25 ug/ml RA and 5 ng/ml TGF-β₁. Real time quantitive RT-PCRand Westemblot detected the expression of Vimentin and E-cadherin from the mRNA and protein levels.Results: In negative control groups and RA groups, the expressions of Vimentin and E-cadherin had no differences. Compared to the control groups, the expression of Vimentin significantly increased, the expression of E-cadherin significantly decreased in the TGF-β₁ treated groups. Compared to the TGF-β₁ treated groups, RA could inhibit the expression of Vimentin, E-cadherin mRNA expression and proteinproduction increased.Conclusion: RA could block EMT triggered by TGF-β₁, which implies that RAcould participate in renal interstitial fibrosis as a negative regulator. Partâ…¡: Effect of rosmarinic acid on mitochondria during the process of EMTObjective: To investigate the effect of rosmarinic acid on mitochondria duringthe process of EMT induced by transforming growth factor-Pi (TGF-β₁).Methods: Growth-arrested and synchronized mouse proximal tubular cells (mPTCs) were stimulated with TGF-Pi in the presence or absence of RA. Cultured cells were divided into 4 groups: A. negative control; B. 25 ug/ml RA; C. 5 ng/ml TGF-β₁;D. 25 ug/ml RA and 5 ng/ml TGF-β₁. The mitochondrial DNA (mtDNA) copy number was evaluated by real time quantitiveRT-PCR Cellular ATP production was determined using luciferase-based luminescence assay. The mitochondrial membrane potential (△Ψm) was detected by JC-1 and reactive oxygen species (ROS) by DCFDA (2',7'-Dichlorofluorescein diacetate, DCFDA) with fluorescence microscope and flow cytometry.The activity of succinate dehydrogenase (SDFI) was detected by chemocolorimetry method. The Mn superoxide dismutase (SOD2), and Peroxisome proliferator-actived receptorγ(PPARγ) coactivator la (PGC-1α) mRNA levels were measured by real time quantitiveRT-PCR,and the protein was detected by western blot.Results: (1) All indicators had no significant differences in the negative control groups and RA groups. (2) TGF-β₁ dramatically increased the cellular ROS levels, RA could cut down the level. (3) The mtDNA in TGF-β₁ treated groups was significantly lower than that the control groups, and RA could increase the expression of mtDNA. (4) Total cellular ATP was dramatically decreased in TGF-Pi treated groups, which was recovered when the cells treated together with the RA. (5) RA could partially restore the reduced activity of succinate dehydrogenase caused by TOF-β₁. (6) The mitochondrial membrane potential of TGF-β₁-treated cells tended to decrease, and RA would reinstate this. (7) Compared to the control groups, the expression of SOD2, PGC-1αmRNA and protein increased, which were all recovered when cultured with RA. Conclusion: The TGF-β₁ can cause mitochondrial dysfunction in the process of EMT, RA should partially inhibit EMT triggered by TGF-β₁ via protect the mitochondria in the mPTCs.