Protective Effects And Mechanisms Of Tribulus Terrestris Saponin Monomer B On Rat Hearts Impaired By Ischemia/reperfusion

Thrombotic occlusion of a coronary artery often leads to myocardial ischemia, and causes depression of myocardial function and associated deleterious morphologic alterations that lead to heart failure. Re-establishment of blood flow to the ischemic region is essential to salvage ischemic myocardium, and it is generally performed by thrombolysis, percutaneous coronary intervention, or coronary artery bypass grafting. Unfortunately, reperfusion to restore blood flow to the site of injury also appears to facilitate a series response and can cause the same or even more serious injury than only ischemia, which is called ischemia-reperfusion (I/R) injury. Ischemia/reperfusion (IR)-injury is one of the most common pathophysiological processes after acute myocardial infarction, heart stroke, cardiopulmonary bypass surgery, and heart transplantation. The combination of ischemia and subsequent reperfusion injury (I/R injury) can result in additional damage to the heart and oxidants might play key role in inducing myocardial damage. The most potent mechanism of protection on I/R is ischemic preconditioning, a condition in which short episodes of ischemia/reperfusion activate survival kinases that will protect the heart against myocardial infarction during subsequent ischemia. However, it is a mechanism mechanical damage to the body and have difficult to investigate apoptosis in myocardial ischemia /reperfusion (I/R) injury and to find effective drugs to prevent it.Tribulus terrestris is a traditional Chinese plant which has many activitied. Gross saponins from Tribulus terrestris (GSTT) include spiral vagina steroid and snail steroid and are the major derivatives of Tribulus terrestris. GSTT is a well-known Chinese medicine used for the treatment of various diseases including hypertension, hyperlipidemia, platelet aggregation, and aging. The mechanisms by which GSTT protects the ischemic heart have been extensively investigated in our laboratory for many years. Moreover, some studies have proven that GSTT has a significant protective effect against ischemia/reperfusion injury in rat hearts and brains in vitro and in vivo. However, up to now, little is known about which specific component in GSTT plays the important role.We have identified nine saponin monomers and named them A~I transitorily. Each of them is a kind of spiral vagina steroid. So the aim of this study is to detect their bio-activity and elucidate the mechanisms for the protective effect of them on cardiomyocytes.The study is carried by following several parts. In the first study, we observe protection effect of GSTT on cadiocytes impaired by adriamycin and approach the dose in optimization and as it a positive drug in the future research.1.Effect of gross saponins of Tribulus terrestris on cadiocyte impaired by adriamycinCadiocytes of neonate rat were cultivated 72 hours and divided into normal control group, adriamycin (ADR) at 2 mg·L-1 group, and GSTT at 100,30,10 mg·L-1 group. Cadiocyte survival rate was detected with MTT colorimetric method. Contents of CK, LDH, AST, SOD,MAD and NO were detected. Apoptosis was detected with flow cytometry and Caspase-3 expression was detected with western blotting.Compared with control group, contents of CK, LDH, AST, MAD and NO increased and activity of SOD reduced in ADR group(P<0.05,P<0.01). While treated with GSTT 100,30 mg·L-1 , numbers of survival cells increased(P<0.05, P<0.01), contents of CK, LDH, AST, MAD, and NO decreased, and activity of SOD increased(P<0.05, P<0.01). Apoptosis and expression of Caspase-3 also reduced in GSTT 100,30mg·L-1 group. GSTT can protect cadiocytes impaired by ADR, which are possible involved in its effect of resisting oxygen free radical. It has best effect at a dose of 100 mg·L-1.2. Bio-activety screening of TTSMNine monomers of spirostanol saponins were isolated and idendified as JA～JI(named transitorily ) by means of NMR spectrometry. After bio-activity screening on them deploy with MTT, we defined that monomers tigogenin 3–O–β-D-xylopyranosyl(12)-[β- D-xylopyranosyl(1→3)]-β-D-glucopyranosyl (1→4)–[α–L-rhamnopyranosyl(1→2)]–β-D-galactopyranoside (compound JB) and Hecogenin3-O-β-D- glucopyranosyl ( 1→4)–β-D-galactopyranoside (compound JG) have cytoprotective bio-activity. JB display effective dose in 10-9~10-7 mol?L-1, and JG in 10-9 to10-6 mol?L-1. For the yield of JB is high, we research the effect of JB.3. Protective effects of Tribulus terrestris saponin monomer B on rat hearts impaired by ischemia/reperfusion(1)TTSMB protects rat hearts from ischemia/reperfusion injury Isolated rat hearts subjected to 30 min of ischemia followed by 120 min of reperfusion using Langendorff's technique were assigned to the following groups: control, ischemia/reperfusion (I/R), treatment with gross saponins from Tribulus terrestris (GSTT) 100 mg/L, treatment with TTSMB (100, 10, and 1 nmol/L) and treatment with a PKC inhibitor (chelerythrine). Infarct size was assessed by triphenyltetrazolium chloride staining. Malondialdehyde (MDA), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) contents as well as superoxide dismutase (SOD) and creatine kinase (CK) activities were determined after the treatment. Histopathological changes in the myocardium were observed using hematoxylin–eosin (H&E) staining. Apoptosis was detected with terminal deoxynucleotidyl transferase nick-end labeling TUNEL assay. Bcl-2, Bax, Caspase-3 and PKCεprotein expression were examined using western blotting. The results show that TTSMB treatment reduced MDA, AST, CK and LDH contents and increased the activity of SOD. TTSMB treatment reduced the cardiac infarct size and myocardial apoptosis rate. Bcl-2 and PKCεprotein expression increased after TTSMB preconditioning, whereas Bax and Caspase-3 expression decreased. In the chelerythrine group, Bcl-2 and PKCεexpression decreased, whereas Bax and Caspase-3 expression increased.TTSMB has protective effect against myocardial ischemia/reperfusion injury, and PKCεactivation is the underling mechanism.(2)Protective effect of Tribulus terrestris saponin monomer B on cadiocytes impaired by hypoxia-reoxygenationWe observe protection effect of Tribulus terrestris saponin monomer B (TTSMB) on cadiocytes impaired by hypoxia-reoxygenation (H/R), which can mimic the I/R-injury process in vivo. Cadiocytes of neonate rat were cultivated 72 hours and divided into normal control group, H/R group, GSTT at 100 mg·L-1 group and TTSMB at 10,1,0.1 nmol·L-1 group. Morphocytology change of cadiocytes was observed after the treatment. Cadiocyte survival rate was detected with MTT colorimetric method. Levels of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), malondialdehyde (MDA), and activity of superoxide dismutase (SOD) were determined. Apoptosis rate was detected with flow cytometry. Expression of Caspase-3 were examined with western blotting.Compared with control group, survival cell numbers in H/R group decreased obviously, content of CK, LDH, AST, MAD increased, and activity of SOD decreased(P<0.01 and P<0.001). Compared with model group, survival cell population increased in TTSMB 10,1,0.1 nmol·L-1 group(P<0.05 and P<0.01). Contents of CK, LDH, AST, MAD decreased, whereas activity of SOD increased(P<0.05 and P<0.01). Apoptosis rate and expression of Caspase-3 also reduced in TTSMB 10,1,0.1 nmol·L-1 group.TTSMB can against oxygen free radical, inhibit cadiocyte apoptosis and has significant cadiocytes protective effect against H/R.(3)Mechanisms of TTSMB protect rat hearts from H/R injury TTSMB, a component of Gross saponins of Tribulus terrestris (GSTT), has been shown to produce cytoprotective effects in heart. Yet, the precise mechanisms are not fully understood. In order to shed light on this issue, ventricular myocytes were isolated from neonatal rat hearts and were exposed to 3 h of hypoxia followed by 2 h reoxygenation.Apoptosis was induced by hypoxia/reoxygenation (H/R) and the expression of PKCεand ERK1/2 in cultured neonatal rat cardiocytes were detected.The results indicated that treatment with TTSMB protected cardiocytes against apoptosis induced by hypoxia/reoxygenation. PKCεand ERK1/2 expression increased after pretreated with TTSMB. In the presence of PKCεinhibitor to induce preconditioning, the expression of ERK1/2 was decreased in hypoxia/reoxygenationed cardiocytes co-treated with TTSMB. While preconditioned with PD98059, no effects on expression of PKCεwere detected..In conclusion, TTSMB has protective effects on cardiocytes against apoptosis induced by hypoxia/reoxygenation injury via PKCεand ERK1/2 signaling pathway. The results of the present study, we arrived at the following concludes:After preliminary screening, we determined that TTSMB whose chemical name is tigogenin3-0-β-D-xylopyranosyl(1-2)-[β-D-xylopyranosyl(1-3)]-β-D-glucopyranosyl(1-4)-[a-L-rhamnopyranosyl(1-2)]-β-D-galactopyranoside, also called tribulosin, is bioactive.TTSMB has a protective effect against myocardial ischemia/reperfusion injury, and which can against oxygen free radical and inhibit cadiocyte apoptosis, and has shown to produce cytoprotective effects on cardiomyocytes. The effects on cardiocytes against apoptosis induced by hypoxia/reoxygenation injury via PKCεand ERK1/2 signaling pathway.