| Background:Diseases are considered to be resulted from multiple factors. Western medicine, derived from single-target development, can not achieve satisfied therapeutic effects because of the limitation, whereas, the advantage and unique characteristics of Traditional Chinese Medicine(TCM) lie in multi-target therapeutic strategies. For thousands of years, the therapeutic model of multi-target and overall treatment of complicated TCM has been established by the earliest record of “Shennong tasted hundreds of grasses”. Western medicine has followed this model and multi-drug combination of comprehensive treatment has been gradually perfromed in clinic. However, comparing to western medicine, the biggest issue of TCM is “blinding treatment”, including unknownness of active substances, therapeutic targets and the law of compatibility of medicines in TCM prescriptions, which has seriously impeded the process of internationalization of TCM. Recent researches indicate that effective components compatibility is a novel strategy forthe application of TCM, which simplified the formula and is helpful for the modernization of connotation in TCM. Therefore, it has important significance theoretically to seek for the effective components in prescriptions, with the help of modern molecular targets and detection technology. Thus, the connotation mystery in the compatibility of TCM prescriptions will be uncovered and the essence of TCM will be inherited and carried forward to promote the internationalization of TCM.The theory of TCM considers the evil of all diseases lies in blood stasis which can cause blood thickness, viscousness, aggregation and coagulation. When blood stasis happens in the heart, myocardial ischemia would be induced; when blood stasis happens in the kidney, renal ischemia would be induced. The lethal blood reperfusion always follows the treatment of myocardial ischemia. Therefore, secondary myocardial ischemia/reperfusion(MI/R) injury has been increasingly paid high attention. The core of therapeutic strategies of TCM should be to activate blood circulation to remove blood stasis. Similarly, renal ischemia for a long time leading to insufficient blood perfusion would progress into chronic kidney injury(CKI). It is regarded as “Longbi, Guan’ge, Nidu” in TCM, in which blood stasis is the most common syndrome. The characteristic pathogenesis is qi stagnation and blood stasis which result in the derivation of turbid toxic material. Therefore, “activating circulation, releasing stasis, and draining turbidity detoxification” is believed as a therapeutic principle for CKI in TCM. Thus it can be seen that promoting circulation and removing stasis is the fundamental therapeutic principle in either MI/R injury or CKI.Since the ancient times, TCM has been the only way to treat diseases resulted from blood stasis. Salvia miltiorrhiza is a kind of traditional herb medicine functioned as promoting circulation and removing stasis, with which there are a huge number of medicines to be compatible. For instance, Danhong Injection(DHI), a modern compound preparation comprised of Salvia miltiorrhiza and Safflower carthamus, is mainly prescribed in the treatment of cardiovascular and cerebrovascular diseases. For another instance, there are dozens of Chinese patent medicines and formulas(for example, Shenkang injection, Shenshuaining capsule, and Uremic clearance granule, et al) for thetreatment of CKI, which mainly comprised of Salvia miltiorrhiza and Chinese rhubarb.However, until now, there is still unclear about the connotation and the molecular mechanisms in cardioprotection and renoprotection of Salvia miltiorrhiza pair-herb compatibility, which seriously impedes the quality improvement and secondary development of effective prescriptions. So it plays a pivotal role in quality improvement of large varieties, inheritance and promotion of the essence, and advancement of the modernization and internationalization of TCM, to elucidate the active substances and mechanisms in Salvia miltiorrhiza pair-herb compatibility.In this study, based on the research of Salvia-Safflower pair-herb application in MI/R injury and Salvia-Rhubarb pair-herb application in CKI, we try to seek and verify the active substances and mechanisms of Salvia miltiorrhiza pair-herb, to investigate molecular mechanisms in Salvia miltiorrhiza pair-herb and to uncover the scientific connotation of pair-herb. This study might be helpful to provide some new meaningful messages and references for the pair-herb and prescriptions research in the future.Objectives:Part 1: Active substances and mechanisms of Salvia-Safflower pair-herb for MI/R injury improvement.1. Screening of active substances for cardioprotection in DHI and verification by feedback test.2. Exploration of the cardioprotection of DHI and its active substances, including the effects of anti-imflammation, anti-oxidative stress, and anti-apoptosis.3. Confirmation the mechanism of DHI and its active substances through the activation of reperfusion injury salvage kinase(RISK) signaling pathway to improve MI/R injury.Part 2: Active substances and mechanisms of Salvia-Rhubarb pair-herb for CKI improvement.1. Screening of active substances in Salvia-Rhubarb pair-herb and verification by feedback test.2. Exploration of the renoprotection of danshensu and rhein combination, including the effects of anti-fibrosis, anti-inflammation and anti-apoptosis.3. Confirmation the mechanisms of danshensu and rhein combination through the activation of Sirt3/FOXO3 a signaling pathway to improve CKI.Methods:Part 1: Active substances and mechanisms of Salvia-Safflower pair-herb for MI/R injury improvement.1. Active substances screening: Cardiomyocyte model induced by simulated ischemia/reperfusion(SI/R) injury in vitro was biult and administered with the main components, such as danshenshu, salvianolic acid A, salvianolic acid B, rosmarinic acid, protocatechuic aldehyde, hydroxysafflor yellow A, and carthamin. To screen out the candidate active substances in DHI, the cell viability was detected by MTT assay; the release of CK-MB and LDH was detected by ELISA.2. Candidate active substances feedback test and verification: The rat MI/R injury model was built and administered with candidate active substances either individually or in combination. The changes of cardiac function were observed and myocardial infarct size was determined by Evans Blue and TTC staining; the serum levels of CK-MB and c Tn I were detected by ELISA; the injuries of myocardium were observed by H&E staining; the cell viability was conducted by MTT assay; the quantity of LDH in the supernatant was measured by biochemical techniques.3. Anti-inflammation: DHI and active substances were administrated to the MI/R injury rats or SI/R injury primary cardiomyocyte. The inflammation cytokines, including TNF-α, IL-1β, and IL-10 in the serum of rats and the supernatant of cells were detected by ELISA; and the protein expression of NF-kB pathway was analysed by western blot to evaluate their function in anti-inflammation.4. Anti-oxidation: DHI and active substances were administrated to the MI/R injury rats or SI/R injury primary cardiomyocyte. The MDA and SOD level in the serum of rats and the supernatant of cells were detected by biochemical techniques; and the proteinexpression of NF-E2-related factor 2(Nrf2) pathway was tested by western blot to evaluate their function in anti-oxidation.5. Anti-apoptosis: DHI and active substances were administrated to the SI/R injury primary cardiomyocyte. Flow cytometry and Terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling(TUNEL) assay were used to detect the cell apoptosis in vitro; and the activity of Caspase-3, the key enzyme in the apoptotic cascade, was detected by ELISA to evaluate their function in anti-apoptosis.6. Activation of RISK signaling pathway: DHI and active substances were administrated to the SI/R injury primary cardiomyocyte, meanwhile, phosphatidylinositol-3-kinase(PI3K) inhibitor(wortmannin) and extracellular-signal regulated kinase 1/2(ERK1/2) inhibitor(U0126) were used to verify the expression of PI3K/Akt and ERK1/2 pathways protein by western blot in order to investigate their activation of RISK pathway.Part 2: Active substances and mechanisms of Salvia-Rhubarb pair-herb for CKI improvement.1. Active substances screening: HK-2 cell model induced by the transforming growth factor-β(TGF-β) in vitro was built and administered with the main components, such as danshenshu, salvianolic acid A, salvianolic acid B, protocatechuic aldehyde, rosmarinic acid, rhein, emodin, and aloe-emodin. To screen out the candidate active substances in Salvia-Rhubarb pair-herb, the cell viability and the release of inflammatory cytokines were detected; and their optimal proportion was distinguished by MTT assay.2. Candidate active substances feedback test and verification: The rat 5/6 nephrectomy(5/6Nx) model was built to simulate clinical CKI and administrated with candidate active substances individually or in combination for 16 weeks. The body weight of each week, the level of Scr and BUN of every two weeks were monitored; the morphology of residual kidney was evaluated by appearance observation; the kidney blood supply was detected by contrast-enhanced ultrasonography(CEUS), and the ultrastructure of kidney was observed by transmission electron microscope.3. Anti-fibrosis: The active substances were administrated to the 5/6Nx rats and TGF-β induced HK-2 cells individually or in combination. Masson and H&E staining were used to evaluate the fibrosis, glomerulosclerosis index, and renal tubular damage score in residual kidneys; the expression of fibrosis markers in residual kidneys, such as E-cadherin and alpha-smooth muscle actin(α-SMA), were detected by immunohistochemistry staining; the expression of TGF-β/Smad3 pathway and other fibrosis related proteins were detected by western blot, to evaluate their function in anti- fibrosis.4. Anti-inflammation: The active substances were administrated to the 5/6Nx rats individually or in combination. RT-PCR and ELISA assay were used to detect inflammation related cytokines in residual kidneys in the m RNA and cytokine level, including TNF-α, MCP-1, IL-1β, and IL-6; the expression of inflammation pathway related proteins, such as NF-kB, IkBα, ICAM-1, and VCAM-1, were detected by western blot to evaluate their function in anti-inflammation.5. Anti-apoptotsis: The active substances were administrated to 5/6Nx rats and TGF-β induced HK-2 cells individually or in combination. The apoptosis was detected by TUNEL staining in residual kidneys and the cells; the expression of apoptotic related proteins, such as Bax, Bcl-2, and Caspase-3, were detected by western blot to evaluate their function in anti-apoptosis.6. Activation of Sirt3/FOXO3 a signaling pathway: The active substances were administrated to 5/6Nx rats individually or in combination. The expressions of Sirt3 and Ac-FOXO3 a in residual kidneys were detected by immunohistochemistry staining; the expressions of the pathway related proteins in residual kidney tissue homogenate were detected by western blot to evaluate their function in the activation of Sirt3/FOXO3 a signaling pathway. Sirt3 si RNA was transfected into HK-2 cells. The expression of E-cadherin and α-SMA was detected by immunofluorescence staining; the expression of TGF-β/Smad3 pathway was detected by western blot; the inflammation related cytokines and cell apoptosis were analysed to evaluate the key functions of Sirt3 in renoprotection.Results:Part 1: Active substances and mechanisms of Salvia-Safflower pair-herbfor MI/R injury improvement.1. Active substances screening: Five main ingredients in DHI for myocardial protection are screened out by MTT and myocardial enzyme detection: hydroxysafflor yellow A(A), salvianolic acid B(B), danshensu(C), protocatechuic aldehyde(D) and rosmarinic acid(E).2. Candidate active substances feedback test and verify: In MI/R rats, compared with the MI/R group, DHI, A, B, C and the mixture of five candidate active substances above all significantly increased the cardiac function indexes, decreased the infarction sizes, lowered the serum levels of CK-MB and c Tn I, and improved the pathological injuries. Whereas, there were no changes observed in D and E group. Besides, the effectiveness of mixture of the five candidate active substances is equal to DHI’s potency. In SI/R injuried myocardial cells, compared with the SI/R group, DHI, A, B and C all increased the cell survival rates, decreased the LDH activities. The results of injury attenuation in vivo and in vitro confirmed that A, B, and C are the active substances in DHI.3. Anti-inflammation: In either the serum of MI/R injury rat or the supernatant of SI/R injuried primary cardiomyocyte, the levels of IL-1β and TNF-α were significantly decreased and the level of IL-10 was increased with A, B, C, and DHI pretreatment, which exhibited their functions of anti-inflammation. The underlying mechanism lied in the inhibition of NF-κB expression in inflammation pathway. And these effects could be inhibited by the application of wortmannin or U0126. The results of multiple comparisons indicated that the anti- inflammatory effect of A is more potent than that in B and C, but equal to that in DHI.4. Anti-oxidation: In either the serum of MI/R injury rat or the supernatant of SI/R injuried primary cardiomyocyte, the levels of MDA were significantly decreased and the levels of SOD were enhanced with A, B, C, and DHI pretreatment, which exhibitedtheir functions of anti-oxidation. The underlying mechanism lied in the enhancement of Nrf2 expression in oxidative stress pathway. And these effects could be inhibited by the application of wortmannin or U0126. The results of multiple comparisons indicated that the anti-oxidative effect of B was more potent than that in A and C, but equal to that in DHI.5. Anti-apoptosis: In SI/R injuried primary cardiomyocyte, the results of flow cytometry and TUNEL assay indicated that the apoptotic rate was significantly decreased with A, B, C, and DHI pretreatment, which exhibited their functions of anti-apoptosis. The underlying mechanisms lied in the inhibition of Caspase-3 activity. And these effects could be inhibited by the application of wortmannin or U0126. The results of multiple comparisons indicated that the anti-apoptotic effect of C is slightly more potent than that in A and B, but equal to that in DHI.6. Activation of RISK signaling pathway: In SI/R injuried primary cardiomyocyte, the results of western blot indicated that comparing with SI/R group, the expression of p-Akt and p-ERK1/2 were increased with A, B, C, and DHI pretreatment. And these effects could be inhibited by the application of wortmannin or U0126, which demonstrated that DHI and the active substantces protected against MI/R injury through the activation of RISK signaling pathway.Part 2: Active substances and mechanisms of Salvia-Rhubarb pair-herbfor CKI improvement.1. Active substances screening: Two main ingredients of Salvia-Rhubarb pair-herb for renoprotection are screened out by MTT and inflammatory factors detection: danshensu and rhein.2. Candidate active substances feedback test and verify: In 5/6Nx rats after 16 weeks’ administration, danshensu and rhein combination protected against kidney injury and improve the poor growth, damaged renal function and poor ultrastructure, comparing to single use. These results confirmed that danshensu and rhein were the active substances in Salvia-Rhubarb pair-herb.3. Anti-fibosis: In 5/6Nx rats, the fibrosis degress and pathological injury wereattenuated with danshensu and rhein treatment, which exhibited their functions of anti-fibosis. The underlying mechanisms lied in the inhibition of TGF-β/Smad3 pathway activation, increase the expression of E-cadherin and decrease the expressionof α-SMA, Collagen-I, and FN. The anti-fibrotic effect was more potent incombination than single use.4. Anti-inflammation: In 5/6Nx rats, the expressions of TNF-α, MCP-1, IL-1β, and IL-6 in the m RNAs and cytokine levels decreased with danshensu and rhein treatment, which exhibited their functions of anti-inflammation. The underlying mechanisms lied in the inhibition of NF-?B pathway and expression of adhesion molecules related proteins. The anti-inflammatory effect was more potent in combination than single use.5. Anti-apoptosis: In 5/6Nx rats and TGF-β injuried HK-2 cells, the positive rate of TUNEL staining significantly decreased with danshensu and rhein treatment, which exhibited their functions of anti-apoptosis. The underlying mechanisms lied in the decrease of expression of Bax and Caspase-3, and increase of expression of Bcl-2. The anti-apoptotic effect is more potent in combination than single use.6. Activation of RISK signaling pathway: In 5/6Nx rats, the expression of Sirt3 significantly was increased and the ratio of Ac-FOXO3a/FOXO3 a was decreased with danshensu and rhein treatment, which exhibited their functions on activation of Sirt3/FOXO3 a pathway. In Sirt3 si RNA transfected HK-2 cells, the functions of inhibiting fibrosis, inflammation and apoptosis by danshensu and rhein treatment, were all abrogated. These results suggested that Sirt3 might play a pivotal role in renoprotection of danshensu and rhein combination.Conclusions:1. Hydroxysafflor yellow A(A), Salvianolic acid B(B), Salvianic acid(C) are confirmed as the active substances in DHI.2. The three active substances(A, B, and C) and DHI can attenuate MI/R injury.3. The three active substances(A, B, and C) and DHI can suppress the inflammation, oxidative stress and cell apoptosis in MI/R injury.4. The three active substances(A, B, and C) and DHI protect against MI/R injury by the activation of RISK signaling pathway.5. Danshensu and rhein are confirmed as the main active substances in Salvia-Rhubarb pair-herb.6. Danshensu and rhein combination can obtain good therapeutic effect on the treatment of CKI.7. Danshensu and rhein combination can suppress kidney fibrosis, inflammation reaction and cell apoptosis in CKI.8. Danshensu and rhein combination protect against CKI by the activation of Sirt3/FOXO3 a signaling pathway. |
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