| Aims To investigate the expression patterns of galectin-3 and sp1 in the myocardium of SHR with different week ages, and determine the effect of galectin-3 on the differentiation, collagen production, and sp1 expression of myocardial fibroblast (Fb), to explore the potential role and mechanism of galectin-3 on the myocardial fibrosis, providing new insights for the basic and clinic researches of myocardial fibrosis.Methods SHR of 12w, 20w and 28w were applied for the experiments, WKY in the same week-age as control. The blood pressure of caudal artery was detected , the ratio of left ventricular weight to body weight (LVW/BW) was calculated. Masson method was applied for the collagen staining to analyze the collagen volume fraction (CVF) and the ratio of perivascular collagen area to luminal area (PVCA/LA). ELISA was applied to detect PICP concentration in rat serum. The mRNA levels of galectin-3 and sp1 were determined by RT-PCR, the protein levels of galectin-3 and sp1 were determined by western blot, and the protein level of galectin-3 was also determined by immunohistochemistry. The differences between WKY and SHR, and those between different week-age groups of SHR were analyzed by SPSS software.Rat myocardial Fb were cultured with stimulation of galectin-3 in different concentrations (10, 20, 30, 40μg/ml for 24 hours) or different time periods (6, 12, 24, 48 hours in 20μg/ml). The mRNA levels ofα-SMA and sp1 were detected by RT-PCR, and the protein levels were detected by western blot. PICP concentration in culture medium was determined by ELISA. By the transfection of sp1 antisense oligonucleotides, the expressions ofα-SMA and sp1 induced by galectin-3 were determined by RT-PCR and western blot, as well as PICP concentration by ELISA.Results (1) The average blood pressures of SHR of 12w, 20w and 28w was significantly higher than that of age-matched WKY. From the week age of 12 to 28, the blood pressure of SHR was increased gradually, and the average blood pressure of the group in of 20w was much higher than the group of 12w, and the blood pressure of the group of 28w was also higher than the group of 20w. The differences of LVW/BW were consistent with the change of blood pressure. By the histological examination, the obvious myocardial interstitial and perivascular fibrosis presented in the SHR of 20w, and the local fibrosis scar presented in the group of 28w, with the gradually aggravated fibrosis lesion. The image analysis showed that CVF and PVCA/LA of SHR in 20w and 28w were much higher than that of WKY in the corresponding week age. CVF and PVCA/LA of SHR of 28w were significantly higher than that of SHR of 20w; CVF and PVCA/LA of SHR of 20w were drastically higher than that of 12w. Compared with age-matched WKY, PICP concentration of SHR of 20w and 28w were highly increased. The PICP concentration of SHR of 20w was significantly higher than that of SHR of 12w, and the PICP concentration of SHR of 28w was drastically higher than that of 20w.(2) Compared with age-matched WKY, the mRNA and protein levels of galectin-3 of SHR of 20w and 28w were much highly increased. The mRNA and protein level of galectin-3 of SHR of 20w was signifieantly higher than that of SHR of 12w, and the galectin-3 mRNA and protein level of SHR of 28w was drastically higher than that of 20w. As for the immunohistochemistry results, the positive staining was presented in myocardial interstitium of SHR of 20w, and the positive average area density of SHR of 28w was signifieantly higher than that of SHR of 20w.(3) Compared with age-matched WKY, the mRNA and protein levels of sp1 of SHR of 20w and 28w were highly increased. The mRNA and protein level of sp1 of SHR of 20w was signifieantly higher than that of 12w, and the sp1 mRNA and protein level of SHR of 28w was drastically higher than that of 20w.(4) The mRNA and protein levels ofα-SMA in Fb increased obviously in response to galectin-3 stimulation with the concentration of 10μg/ml, which were in a dose-dependent manner. By the stimulation of galectin-3 with the concentration of 20μg/ml, the mRNA and protein levels ofα-SMA were highly increased after 12 hours, and gradually increased until the time point of 48 hours.(5) The serum concentration of PICP in Fb culture medium increased obviously in response to galectin-3 stimulation with the concentration of 20μg/ml, which was in a dose-dependent manner. By the stimulation of galectin-3 with the concentration of 20μg/ml, the serum concentration of PICP was highly increased after 12 hours, and gradually increased until the time point of 48 hours.(6) The mRNA and protein levels of sp1 in Fb increased obviously in response to galectin-3 stimulation with the concentration of 10μg/ml, which were in a dose-dependent manner. By the stimulation of galectin-3 with the concentration of 20μg/ml, the mRNA and protein levels of sp1 were highly increased after 12 hours, and gradually increased until the time point of 48 hours.(7) The mRNA and protein levels of sp1 induced by glactin-3 in sp1 antisense oligonucleotide group (AS) were much lower than the group of ctrl and Inv, as well as the levels ofα-SMA and PICP.Conclusions (1) The obvious myocardial fibrosis presented in SHR of 20w, which was aggravated in SHR of 28w.(2) The expression of galectin-3 was increased in SHR of 20w, which was increased in SHR of 28w further, in accordance with the severity of myocardial fibrosis of SHR.(3) The expression of sp1 was increased in SHR of 20w, which was increased in SHR of 28w further, in accordance with the severity of myocardial fibrosis of SHR and the changes of galectin-3 levels.(4) Exogenetic galectin-3 could induce the differentiation of Fb, and the expression ofα-SMA in Fb in a time- or dose-dependent manner.(5) Exogenetic galectin-3 could induce the collagen production in Fb in a time- or dose-dependent manner.(6) Exogenetic galectin-3 could induce the expression of sp1 in Fb in a time- or dose-dependent manner.(7) The sp1 antisense oligonucleotides could inhibit the endogenous expression of sp1, resulting in the inhibition of differentiation and collagen production of Fb induced by galectin-3. |
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