Network Pharmacology along with Molecular Docking to Explore the Mechanism of Danshen Injection against Anthracycline-induced Cardiotoxicity and Transcriptome Validation.

INTRODUCTION: Although anthracyclines have demonstrated efficacy in cancer therapy, their utilization is constrained by cardiotoxicity. In contrast, Danshen injection (DSI), derived from Salvia miltiorrhiza, has a longstanding tradition of being employed to ameliorate cardiovascular ailments, including anthracycline- induced cardiotoxicity (AIC). Nonetheless, there is a notable dearth of comprehensive systematic investigation into the molecular mechanisms underlying DSI's effects on AIC. Consequently, this study was undertaken to explore the underlying mechanism by which DSI acted against AIC. METHODS: Employing network pharmacology approach, the current investigation undertook a comprehensive analysis of the impact of DSI on AIC, which was further validated by transcriptome sequencing with in vitro AIC model. Additionally, molecular docking was conducted to evaluate the binding of active ingredients to core targets. A total of 3,404 AIC-related targets and 12 active ingredients in DSI, including chrysophanol, luteolin, tanshinone IIA, isoimperatorin, among others, were collected by differentially expressed analysis and database search, respectively. RESULTS: The network pharmacology and enrichment analysis suggested 102 potential targets and 29 signaling pathways associated with the protective effect of DSI on AIC. Three core targets (CA12, NOS3, and POLH) and calcium signaling pathways were further validated by transcriptomic analysis of the in-vitro model. The high affinity of the active ingredients binding to corresponding targets was confirmed by molecular docking. CONCLUSION: The present study suggested that DSI might exert a cardioprotective effect on AIC via the inhibition of CA12, NOS3, and POLH, as well as the modulation of calcium signaling. Further experiments are warranted to verify the findings.

Efficacy of Danshen injection combined with Calcitriol and Calcium/Vitamin-D for the treatment of osteoporotic fractures: A retrospective case-control study.

Objective: To explore the efficacy of Danshen injection combined with calcitriol and calcium/Vitamin-D in the treatment of osteoporotic fractures. Methods: This was a case-control study. We retrospectively reviewed clinical data of 91 patients with osteoporotic fractures who received treatment in Rui'an People's Hospital from February 2021 to July 2022. The data were divided into a control group with 44 records of patients who received treatment with calcitriol and calcium/Vitamin-D, and a study group with 47 patients who received Danshen injection combined with calcitriol and calcium/Vitamin-D. The control group individuals were coordinated with the patients in terms of their age and gender. Treatment effects, inflammatory response levels, and bone metabolic status levels were comparable between the two groups before and after the treatment. Results: The total efficacy of the treatment in the study group was better than that in the control group (P<0.05). After the treatment, levels of serum inflammatory factors in both groups decreased compared to those before the treatment, and the study group displayed lower levels than the control group (P<0.05). After the treatment, the bone metabolism status of both groups improved, and the improvement effect of the study group was better (P<0.05). The incidences of adverse reactions were similar in both groups (P>0.05). Conclusions: Danshen injection combined with calcitriol and calcium/Vitamin-D for the treatment of osteoporotic fractures can effectively reduce inflammation, regulate bone metabolism, and improve fracture treatment efficacy with a favorable safety profile.

Danshen Injection Inhibits Platelets-induced Metastasis of Breast Cancer Cells In Vitro / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danshen injection （DAN） on platelet （PLT）-induced metastasis of breast cancer cells in vitro. MethodThe 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） assay was used to observe the effect of DAN on the growth of MDA-MB-231 cells in vitro. Oris™ migration assay was used to determine the effect of DAN （final mass concentrations 4， 8， 16 g·L-1） on PLT （1.5×1010 cells/L）-induced migration of breast cancer cells in vitro. The effect of DAN on PLT-induced cell invasion was detected by Transwell assay. Immunofluorescence and Western blot were used to detect the effect of DAN on the protein expression associated with PLT-induced epithelial-mesenchymal transition （EMT）. In addition， enzyme-linked immune-sorbent assay （ELISA） was used to determine the effect of DAN （final mass concentrations 4， 8， 16， 32， 64 g·L-1） on the secretion of transforming growth factor-β1 （TGF-β1）. Western blot was used to observe the effect of DAN on the expression of podoplanin （PDPN） protein in MDA-MB-231 cells induced by PLT. ResultCompared with the blank group， the DAN groups （32 and 64 g·L-1） showed decreased A570 （P<0.05， P<0.01）， and there was no significant difference in A570 between DAN groups （4， 8， 16 g·L-1）. Compared with the blank group， the PLT group showed increased cell migration and invasion， while DAN groups significantly inhibited PLT-induced cell migration and invasion. Compared with the blank group， the PLT group showed decreased expression of E-cadherin， while DAN could significantly reverse this effect of PLT. Compared with the blank group， the PLT group showed increased Slug and Snail protein expression （P<0.05， P<0.01）， while DAN significantly reversed Snail protein expression induced by PLT （P<0.05， P<0.01）. The content of TGF-β1 in the PLT group increased （P<0.01）， while the secretion of TGF-β1 induced by PLT decreased in the DAN groups （16， 32， and 64 g·L-1） （P<0.05， P<0.01）， and the secretion of TGF-β1 was not significantly affected in other DAN groups. PDPN protein expression in the PLT group increased （P<0.01）， while DAN could significantly inhibit PLT-induced PDPN expression （P<0.01）. ConclusionDAN can inhibit PLT-induced migration， invasion， and EMT of breast cancer cells. The mechanism may be related to the direct action between breast cancer cells and tumor cells by down-regulating PDPN expression and interfering with PLT and has nothing to do with the effect of TGF-β1 secretion of PLT.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

Development of a comprehensive method based on quantitative 1H NMR for quality evaluation of Traditional Chinese Medicine injection: a case study of Danshen Injection.

OBJECTIVES: This study aimed to establish a rapid and comprehensive method for quantitative determination of complex ingredients in Traditional Chinese Medicine injections. METHODS: A 1H quantitative nuclear magnetic resonance method was developed to simultaneously quantify comprehensive chemical components in Danshen Injection. Multivariate statistical analysis technique was applied to quality evaluation of multiple batches of Danshen injection. KEY FINDINGS: A complete signal attribution to the 1H nuclear magnetic resonance spectrum of Danshen injection was developed and performed for the first time. A total of 32 chemical components were identified from Danshen Injection. Among them, 20 were quantified simultaneously, accounting for up to 80% (w/w) of the total solids and 95% (w/w) of total organic matter, representing success compared to the previous studies. The developed method was further applied to analyze 13 batches of Danshen Injection from three manufacturers to make a realistic analysis. CONCLUSION: It was found that the comprehensive chemical information provides an adequate characterization for quality profiles among different commercial batches of Danshen Injection. The developed method further offered a guarantee for improving the consistency and safety of Traditional Chinese Medicine injections.

Efficacy of Danshen injection iontophoresis in the treatment of retinal vein occlusion / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the efficacy of Danshen injection iontophoresis in the treatment of retinal vein occlusion(RVO).METHODS: The clinical data of 90 patients(90 eyes)with RVO treated in our hospital from January 2020 to December 2021 were analyzed retrospectively, and they were divided into control group and observation group according to treatment methods. A total of 38 patients(38 eyes)in the control group were treated with intravenous Xueshuantong+oral compound Xueshuantong capsule+enteric-coated aspirin, and 52 patients(52 eyes)in the observation group were treated with Danshen injection iontophoresis on this basis. The treatment time of the two groups was 3mo. The clinical efficacy, best corrected visual acuity, retinal condition and hemorheological indexes(whole blood low shear viscosity, fibrinogen)were compared between the two groups before and after treatment.RESULTS: The total effective rate of the observation group was significantly higher than that in the control group(87% vs 58%,P&#x003C;0.05)at 3mo after treatment. The best corrected visual acuity in both groups was better than that before treatment, and the observation group was better than the control group(all P&#x003C;0.05).The circulation time of retinal vein, the relative diameter of retinal vein and the relative area of retinal hemorrhage in the observation group were lower than those before treatment,and the observation group was lower than the control group(all P&#x003C;0.05). The whole blood low shear viscosity and fibrinogen levels in the two groups were lower than those before treatment, and the observation group was lower than those in the control group(all P&#x003C;0.05).CONCLUSION: Danshen injection iontophoresis is effective in the treatment of patients with RVO, which can effectively improve the visual acuity, treat fundus lesions and improve the abnormal blood flow.

The clinical efficacy of compound Danshen injection on acute cerebral infarction and on the changes in the CRP, D-dimer, and IL-6 levels.

OBJECTIVE: To study the clinical curative effect of compound Danshen injection on acute cerebral infarction (ACI) patients and its impact on the CRP, D-dimer, and IL-6 levels. METHODS: 116 patients with ACI admitted to our hospital were randomly placed in an observation group (n=58) or a control group (n=58). The control group received rosuvastatin tablets (10 mg/time, qd) in addition to the standard treatment. The observation group received compound Danshen injection in addition to the standard treatment. The treatment continued for 21 days. The clinical treatment efficacy, the CRP, D-dimer, and IL-6 levels, the NIHSS scores (to evaluate the degree of neurological impairment), the Fugl-Meyer scores (to access patients' motor function), the ADL scores, the sleep quality (the PSQI and AIS scores), and the complication incidence rates were compared between the two groups. RESULTS: After the treatment, the effective rate in the observation group (89.66%) was significantly higher than it was in the control group (74.14%) (P<0.05). After the treatment, the serum CRP, D-dimer, and IL-6 levels in the two groups were lower than they were before the treatment, and the levels were lower in the observation group than they were in the control group (all P<0.05). After the treatment, the NIHSS scores in the observation group were lower than they were in the control group, and the Fugl-Meyer and ADL scores were higher than they were in the control group (all P<0.05). Compared with the control group, the PSQI and AIS scores in the observation group were lower than they were in the control group after the treatment (P<0.05). The severe diarrhea, bedsore, urinary tract infection, liver and kidney function injury, skin allergic reactions and other total adverse reaction incidence rates in the observation group were lower than they were in the control group. CONCLUSION: Rosuvastatin combined with compound Danshen injection is effective in ACI treatment. It is able to effectively improve the clinical symptoms, reduce the incidence of complications, improve the recovery of the IL-6, CRP, and D-dimer levels and enhance patients' sleep quality.

[Systematic review of efficacy and safety of Compound Danshen Injection combined with Western medicine in treatment of vascular dementia].

To evaluate the efficacy and safety of Compound Danshen Injection combined with Western medicine in the treatment of vascular dementia. Databases of Cochrane Library, PubMed, EMbase, CNKI, SinoMed, VIP, Wanfang Data were electronically retrieved for collecting randomized controlled trial(RCT)about vascular dementia treated with Western medicine alone or combined with Compound Danshen Injection from the year of database establishment to January 2020. Two researchers independently screened out li-teratures, extracted data, and evaluated the risk of bias for inclusion in the study. Meta-analysis was conducted using RevMan 5.3 software. A total of 5 RCTs were included, involving 588 patients, with 299 in treatment group and 289 in control group. Meta-analysis results showed that compared with Western medicine alone, Compound Danshen Injection combined with Western medicine was better in the effective rate(RR=1.23,95%CI[1.14,1.33],P<0.000 01), MMSE score(MD=3.54,95%CI[3.01,4.06],P<0.000 01), ADL score(MD=11.49,95%CI[8.05,14.93],P<0.000 01), the level of CRP(MD=-0.72,95%CI[-1.25,-0.20],P=0.007) and the level of IL-6(MD=-7.64,95%CI[-9.65,-5.63],P<0.000 01). Adverse reactions mainly included rash and skin prick, which did not affect the treatment effect. Based on the findings, the combination of Compound Danshen Injection in the treatment of vascular dementia could improve the effective rates, relieve the mental state damage and improve the daily living ability, with mild adverse reactions and a low incidence. However, due to the low quality of the included literatures, high-quality and large-scale randomized controlled trials are needed for further verification.

A randomized controlled study on an integrated approach to prevent and treat re-adhesion after transcervical resection of moderate-to-severe intrauterine adhesions

OBJECTIVES: This study aims to compare the clinical efficacy of an integrated approach to prevent and treat the recurrence of moderate-to-severe intrauterine adhesions (IUA) after hysteroscopic transcervical resection of adhesion (TCRA). METHODS: The study included a total of 70 patients with moderate-to-severe IUAs who underwent TCRA. Patients were randomly divided into two groups: treatment group (n=35) and control group n=35). In the treatment group, patients underwent balloon uterine stent placement and artificial cycle as well as received intrauterine perfusion of Danshen injection and oral Chinese medicine. In the control group, patients underwent balloon uterine stent placement and artificial cycle as well as received hyaluronic acid sodium and intrauterine device (IUD). Follow-up was performed after treatment of uterine cavity, menstruation and pregnancy. RESULTS: After 3 months of treatment, we observed a significantly lower rate of intrauterine re-adhesion (45.71% versus 77.14%, p=0.044) and significantly higher clinical efficiency (82.86% versus 77.14%, p=0.025) in the treatment group than those in the control group. After 6 months of treatment, we observed a significantly higher clinical efficiency in the treatment group than that in the control group (88.57% versus 68.57%, p=0.039). During the follow-up period, the pregnancy rate was 45.71% and 37.14% in the treatment group and control group, respectively, although the difference was not statistically significant (p=0.628). CONCLUSIONS: After surgical management of IUA, the integrated treatment combining a uterus stent placement and artificial cycle with Danshen injection and oral Chinese medicine can improve the condition of menstruation, and prevent and treat recurrence of IUA.

Danshen Injection Inhibits SKOV3 Cell Proliferation in Vitro by Interfering with Their Interaction with Platelets / 中国实验方剂学杂志

Objective:To investigate the inhibitory effects of Danshen injection against ovarian cancer cell proliferation induced by the interaction between platelets and cancer cells. Method:The induction of platelets on SKOV3 growth <italic>in vitro</italic> and the inhibitory effect of Danshen injection at 12，24，and 48 g·L^-1 were observed by 3-（4，5-Dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） and cell colony formation assays. The content of transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in the platelet-tumor cell interaction system and platelet supernatant and the effect of Danshen injection on TGF-<italic>β</italic>₁secretion were detected by enzyme-linked immunosorbent assay （ELISA）. The influences of tumor cell culture supernatant on platelet aggregation and secretion and the inhibitory effect of Danshen injection were determined by microplate assay and ELISA. The effects of Danshen injection on platelet nuclear factor kappa B （NF-<italic>κ</italic>B） signaling pathway were assayed by Western Blot. Result:Compared with the blank group， the platelet induction group exhibited significantly elevated absorbance at <italic>A</italic>₅₇₀（<italic>P</italic><0.01）， while the absorbance at <italic>A</italic>₅₇₀ in the platelet + Danshen injection group was significantly lower than that in the platelet induction group （<italic>P</italic><0.01）. The comparison with the Danshen injection group revealed that the cell proliferation inhibitory rate in the platelet + Danshen injection group at the same dose was more significant （<italic>P</italic><0.01）. The number of colonies in the platelet induction group was obviously increased in contrast to that in the blank group（<italic>P</italic><0.05）， while the number of colonies in the platelet + Danshen injection group was significantly lower than that in the platelet induction group（<italic>P</italic><0.05，<italic>P</italic><0.01）. As demonstrated by comparison with the blank group， TGF-<italic>β</italic>₁content in the supernatant of the platelet induction group rose remarkably（<italic>P</italic><0.01）， whereas that in the platelet + Danshen injection group declined（<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the platelet + Danshen injection group displayed more obvious inhibition（<italic>P</italic><0.01）. Compared with the blank group， Danshen injection significantly reduced the TGF-<italic>β</italic>₁content in platelet supernatant（<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant change in the content of TGF-<italic>β</italic>₁in SKOV3 supernatant treated with Danshen injection. The platelet aggregation， thromboxane A₂（TXB₂）， and serotonin （5-HT） secretion in the SKOV3 cell supernatant induction group were significantly increased as compared with those in the blank group （<italic>P</italic><0.01）， while such indexes in the cell supernatant induction + Danshen injection group were obviously decreased （<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the cell supernatant induction + Danshen injection group displayed more obvious inhibition at the same dose（<italic>P</italic><0.01）. Compared with the blank group， the platelet induction group exhibited obviously up-regulated phosphorylated TGF-<italic>β</italic>-activated kinase-1 （TAK-1） and NF-<italic>κ</italic>B， but down-regulated phosphorylated inhibitory protein of NF-<italic>κ</italic>B （I<italic>κ</italic>B）（<italic>P</italic><0.01）， which however were significantly reversed in the platelet + Danshen injection group<bold>（</bold><italic>P</italic><0.01）. Conclusion:Danshen injection affect the proliferation of SKOV3 cells by inhibiting their interaction with platelets， which may be related to the inhibited secretion of TGF-<italic>β</italic>₁.

Systematic review of efficacy and safety of Compound Danshen Injection combined with Western medicine in treatment of vascular dementia / 中国中药杂志

To evaluate the efficacy and safety of Compound Danshen Injection combined with Western medicine in the treatment of vascular dementia. Databases of Cochrane Library, PubMed, EMbase, CNKI, SinoMed, VIP, Wanfang Data were electronically retrieved for collecting randomized controlled trial(RCT)about vascular dementia treated with Western medicine alone or combined with Compound Danshen Injection from the year of database establishment to January 2020. Two researchers independently screened out li-teratures, extracted data, and evaluated the risk of bias for inclusion in the study. Meta-analysis was conducted using RevMan 5.3 software. A total of 5 RCTs were included, involving 588 patients, with 299 in treatment group and 289 in control group. Meta-analysis results showed that compared with Western medicine alone, Compound Danshen Injection combined with Western medicine was better in the effective rate(RR=1.23,95%CI[1.14,1.33],P<0.000 01), MMSE score(MD=3.54,95%CI[3.01,4.06],P<0.000 01), ADL score(MD=11.49,95%CI[8.05,14.93],P<0.000 01), the level of CRP(MD=-0.72,95%CI[-1.25,-0.20],P=0.007) and the level of IL-6(MD=-7.64,95%CI[-9.65,-5.63],P<0.000 01). Adverse reactions mainly included rash and skin prick, which did not affect the treatment effect. Based on the findings, the combination of Compound Danshen Injection in the treatment of vascular dementia could improve the effective rates, relieve the mental state damage and improve the daily living ability, with mild adverse reactions and a low incidence. However, due to the low quality of the included literatures, high-quality and large-scale randomized controlled trials are needed for further verification.

[UPLC fingerprint combined with quantitative analysis of multi-components by single marker for quality assessment of Danshen Injection].

Testing and analysis of chemical markers is currently the prevailing approach for quality assessment of traditional Chinese medicines. However,several important issues remain to be addressed,including the trade-off between accuracy and coverage. In this study,in order to give full play to the advantages of their respective methods and provide technical support for more comprehensively and rapidly evaluate the quality of Danshen Injection products,a fingerprint method was coupled with quantitative analysis of multicomponents by single marker( QAMS),with Danshen Injection as the carrier. Ultra performance liquid chromatography( UPLC) was used to establish the quantitative fingerprint. The UPLC fingerprints contained 13 common peaks,11 of which were identified by using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS). Furthermore,with sodium danshensu as the internal reference substance,relative correction factors( RCFs) of protocatechuic aldehyde,caffeic acid,rosmarinic acid,lithospermic acid,and salvianolic acid B were calculated through slope analysis method,and the QAMS method was adopted to determine the contents of these 6 components. The UPLC fingerprint was employed to assess the consistency of 12 batches of Danshen Injection,which showed good batch-to-batch consistency with similarity higher than 0. 99. In the comparison of contents of the six constituents obtained by QAMS and external standard method( ESM),RSD was all less than 4. 3%,indicating the good accuracy of the QAMS method. The QAMS method developed in this study combined with UPLC fingerprint can comprehensively reflect the internal quality of Danshen Injection when only the reference substance sodium danshensu is consumed,with greatly reduced detection cost and time. It provides a technical basis for further improving the quality standard of Danshen Injection.

Pre-treatment with a combination of Shenmai and Danshen injection protects cardiomyocytes against hypoxia/reoxygenation- and H2O2-induced injury by inhibiting mitochondrial permeability transition pore opening.

Increasing evidence has indicated that opening of the mitochondrial permeability transition pore (mPTP) has a vital role in myocardial ischemia/reperfusion (I/R) injury. Shenmai injection (SMI) plus Danshen injection (DSI) combination, termed Yiqi Yangyin Huoxue (YYH) therapy is used in the clinic to treat cardiovascular diseases, including myocardial I/R injury. Previous studies by our group have demonstrated the protective effect of pretreatment with YYH against myocardial I/R injury in isolated rat hearts. The present study aimed to examine the protective effect of YYH against hypoxia/reoxygenation (H/R)- and H2O2-induced cardiomyocyte injury, and to determine whether this effect is produced by inhibition of mPTP opening. Primary cardiomyocytes isolated from neonatal rats were cultured and randomly grouped into a control group, injury group and pretreatment group, with six duplicated wells in each group during each assay. Cardiomyocytes in the injury group were subjected to H/R to simulate I/R or exposed to H2O2 for 2 h to induce oxidative injury. Cellular injury was assessed via release of creatine kinase (CK) and lactate dehydrogenase (LDH), and cell viability was measured by an MTT assay. The mitochondrial membrane potential (ΔΨm) and cytosolic reactive oxygen species (ROS) were detected using the fluorescent probes rhodamine123 (Rh123) and chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), respectively. Intracellular Ca2+, mitochondrial Ca2+ and mPTP opening were measured using fluo-4 acetoxymethyl (Fluo-4/AM), rhodamine-2 acetoxymethyl (Rhod-2/AM) and calcein acetoxymethyl (Calcein/AM) probes, respectively. The results indicated that pretreatment with YYH enhanced cell viability, increased ΔΨm, reduced CK and LDH release, and decreased intracellular ROS and Ca2+, thus reducing cardiomyocyte injury induced by H/R or H2O2. LY294002, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and PD98059, a specific inhibitor of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway, eliminated the protective effects of the combination therapy on cell viability and the change in the ΔΨm in cardiomyocytes. In conclusion, pre-treatment with YYH has cardioprotective effects against H/R injury and oxidative stress via activation of the PI3K/Akt and Erk1/2 signaling pathways, which reduces mPTP opening, overproduction of ROS and calcium overload.

UPLC fingerprint combined with quantitative analysis of multi-components by single marker for quality assessment of Danshen Injection / 中国中药杂志

Testing and analysis of chemical markers is currently the prevailing approach for quality assessment of traditional Chinese medicines. However,several important issues remain to be addressed,including the trade-off between accuracy and coverage. In this study,in order to give full play to the advantages of their respective methods and provide technical support for more comprehensively and rapidly evaluate the quality of Danshen Injection products,a fingerprint method was coupled with quantitative analysis of multicomponents by single marker( QAMS),with Danshen Injection as the carrier. Ultra performance liquid chromatography( UPLC) was used to establish the quantitative fingerprint. The UPLC fingerprints contained 13 common peaks,11 of which were identified by using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS). Furthermore,with sodium danshensu as the internal reference substance,relative correction factors( RCFs) of protocatechuic aldehyde,caffeic acid,rosmarinic acid,lithospermic acid,and salvianolic acid B were calculated through slope analysis method,and the QAMS method was adopted to determine the contents of these 6 components. The UPLC fingerprint was employed to assess the consistency of 12 batches of Danshen Injection,which showed good batch-to-batch consistency with similarity higher than 0. 99. In the comparison of contents of the six constituents obtained by QAMS and external standard method( ESM),RSD was all less than 4. 3%,indicating the good accuracy of the QAMS method. The QAMS method developed in this study combined with UPLC fingerprint can comprehensively reflect the internal quality of Danshen Injection when only the reference substance sodium danshensu is consumed,with greatly reduced detection cost and time. It provides a technical basis for further improving the quality standard of Danshen Injection.

Protective effects of Danshen injection against erectile dysfunction via suppression of endoplasmic reticulum stress activation in a streptozotocin-induced diabetic rat model.

BACKGROUND: Erectile dysfunction (ED) is a common complication of diabetes. This study aimed to explore the beneficial effect of Danshen injection on ED in a streptozotocin (STZ)-induced diabetic rat model and the underlying mechanism. METHODS: The diabetic rat model was established by an intraperitoneal injection of 60 mg/kg STZ in male Sprague-Dawley rats. The diabetic rats were intraperitoneally injected with Danshen solution (0.5 or 1 mL/kg/day) or the same volume of saline for 6 weeks. Age-matched rats served as controls. After 6 weeks, erectile function and histological morphology of the corpora cavernosum were assessed. Oxidative stress indicators, including superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) levels, were measured in penile tissues. The expression levels of glucose-regulated protein 78 (Grp78), growth arrest and DNA damage-inducible gene 153 (GADD153/CHOP) were determined by immunohistochemistry, immunoblotting, and RT-PCR. Apoptosis was detected by a TUNEL assay. RESULTS: The erection times of diabetic rats were significantly less than those of control rats. Danshen injection could improve erectile function via increased erection times. Danshen injection was also found to ameliorate the morphological abnormalities of the corpora cavernosum, to reduce the number of apoptotic cells, and to suppress caspase-3 activation in penile tissue, accompanied by downregulation of the endoplasmic reticulum stress biomarkers Grp78 and CHOP. Danshen injection could increase SOD activity as well as reduce ROS and MDA levels in diabetic rats, indicating suppression of oxidative stress. CONCLUSION: Danshen injection could rescue diabetes-associated ED, possibly via suppressing the oxidative stress and endoplasmic reticulum (ER) stress-induced apoptosis pathways.

Effects of compound Danshen injection combined with magnesium sulfate on serum MPO and hs-CRP in patients with severe preeclampsia.

Effects of compound Danshen injection combined with magnesium sulfate on serum myeloperoxidase (MPO) and hypersensitive C-reactive protein (hs-CRP) in patients with severe preeclampsia (PE) were investigated. Five hundred patients with severe PE were randomly divided into early-onset observation, early-onset control, late-onset observation and late-onset control groups. Control group was treated with magnesium sulfate, while patients in observation group were treated with magnesium sulfate combined with compound Danshen injection. Serum levels of MPO and hs-CRP were measured by enzyme-linked immunosorbent assay (ELISA) and turbidimetric assay. The effects of compound Danshen injection combined with magnesium sulfate on the above indexes were observed. Serum levels of MPO and hs-CRP significantly decreased in early-onset observation, late-onset observation, early-onset control and late-onset control groups after treatment (p<0.05). After treatment, levels of MPO and hs-CRP were significantly lower in early-onset observation group than in early-onset control group (p<0.05), and levels of MPO and hs-CRP were also significantly lower in late-onset observation group than in late-onset control group (p<0.05). Total effective rate of early-onset observation group and late-onset observation group were higher than that of early-onset control group and late-onset control group. Compound Danshen injection combined with magnesium sulfate achieved better treatment outcomes in the treatment of severe PE than magnesium sulfate alone. The combined treatment can effectively reduce the serum levels of MPO and hs-CRP.

The Efficacy of Danshen Injection as Adjunctive Therapy in Treating Angina Pectoris: A Systematic Review and Meta-Analysis.

BACKGROUND: During the last 40 years, Danshen injection has been widely used as an adjunctive therapy for angina pectoris in China, but its efficacy is not yet well defined. The objective of this study was to verify the efficacy of Danshen injection as adjunctive therapy in treating angina pectoris. METHODS: The major databases including PubMed, Cochrane Library, Sino-Med, Medline, Embase, Google Scholar, China National Knowledge Infrastructure, Wanfang Databases, Chinese Scientific Journal Database, Chinese Biomedical Literature Database and the Chinese Science Citation Database were systematically searched for the published randomised controlled trials (RCTs) on Danshen injection until April 2016. Meta-analysis was conducted on the primary outcomes (i.e., the improvements in symptoms and electrocardiography (ECG)). The quality of the included RCTs was evaluated with the M scoring system (the refined Jadad scale). Based on the quality, year of publication and sample size of RCTs, sensitivity analysis and subgroup analysis were performed in this study. RESULTS: Ten RCTs, including 944 anginal patients, were identified in this meta-analysis. Compared with using antianginal agents (ß-blockers, calcium antagonists, nitrates, etc.) alone, Danshen injection combined with antianginal agents had a better therapeutic effect in symptom improvement (odds ratio [OR], 3.66; 95% confidence interval [CI]: 2.50-5.36) and in ECG improvement (OR, 3.25; 95% CI: 1.74-6.08). CONCLUSIONS: This study showed that Danshen injection as adjunctive therapy seemed to be more effective than antianginal agents alone in treating angina pectoris. However, more evidence is needed to accurately evaluate the efficacy of Danshen injection because of the low methodological quality of the included RCTs.

Effect of Danshen injection on expression of brain-derived neurotrophic factor and insulin-like growth factor-1 in rats with spinal cord injury / 上海交通大学学报（医学版）

Objective: To explore the mechanism of Danshen injection on promoting spinal cord functional recovery by observing the expression of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) in rats with spinal cord injury (SCI). Methods: Thirty SD rats were divided randomly into normal control group, model group and treatment group (n=10). The rats in model group and treatment group were subjected to weight-drop SCI according to the Allens' method, and administered normal saline and Danshen injection, at the dosage of 1 mL/kg once a day, by intraperitoneal injection respectively for 14 days. The combine behavioral score (CBS) of rats in all groups was detected on 1, 3, 7 and 14 days after SCI, the expression of BDNF and IGF-1 in all groups was detected with immunohistochemical method and Western blotting. Results: Compared with the normal control group, the CBS in model group and treatment group was elevated after SCI, and declined gradually with the lapse of injury time in different rates. Until 14 days after SCI, compared with the model group, the CBS in treatment group was lower (P=0.000), and the expression levels of BDNF and IGF-1 (including immunohistochemistry positive cell number and relative content) in treatment group were both elevated significantly (all P<0.05). Conclusion: Danshen injection can promote the recovery of nerve function by elevating the expression of BDNF and IGF-1 after SCI.

Effect of Danshen injection on expression of brain-derived neurotrophic factor and insulin-like growth factor-1 in rats with spinal cord injury / 上海交通大学学报（医学版）

Objective·To explore the mechanism of Danshen injection on promoting spinal cord functional recovery by observing the expression of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) in rats with spinal cord injury (SCI). Methods·Thirty SD rats were divided randomly into normal control group, model group and treatment group (n=10). The rats in model group and treatment group were subjected to weight-drop SCI according to the Allens' method, and administered normal saline and Danshen injection, at the dosage of 1 mL/kg once a day , by intraperitoneal injection respectively for 14 days. The combine behavioral score (CBS) of rats in all groups was detected on 1, 3, 7 and 14 days after SCI, the expression of BDNF and IGF-1 in all groups was detected with immunohistochemical method and Western blotting. Results·Compared with the normal control group, the CBS in model group and treatment group was elevated after SCI, and declined gradually with the lapse of injury time in different rates. Until 14 days after SCI, compared with the model group, the CBS in treatment group was lower (P=0.000), and the expression levels of BDNF and IGF-1 (including immunohistochemistry positive cell number and relative content) in treatment group were both elevated significantly (all P<0.05). Conclusion·Danshen injection can promote the recovery of nerve function by elevating the expression of BDNF and IGF-1 after SCI.

The effect of Danshen extract on lipoprotein-associated phospholipase A2 levels in patients with stable angina pectoris: study protocol for a randomized controlled trial - the DOLPHIN study.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2), a biomarker of oxidation and inflammation, has been associated with increased coronary artery disease risk. To date, very few studies have examined the Chinese herbal drug Danshen or its extract on Lp-PLA2 in patients with stable angina pectoris. In this study, we aim to investigate the effect of Danshen extract on Lp-PLA2 level in patients with stable angina. METHODS/DESIGN: This is a randomized, single-blind, placebo-controlled, adaptive clinical trial. A total of 156 patients meeting the eligibility criteria will be randomly assigned to either the Danshen extract (DanshenDuofensuanyan injection and Danshen drop spill) group or the placebo group in a 1:1 ratio. Participants will then undergo treatment with DanshenDuofensuanyan injection or placebo (glucose) during hospitalization, followed by open-label Danshen drop spill (30 pills/day) in Danshen extract group for 60 days after discharge. Because this is an adaptive trial, two interim analyses are prospectively planned. These will be performed after one-third and two-thirds of the patients, respectively, have completed the trial. On the basis of the results of these interim analyses, a data monitoring committee will determine how to modify aspects of the study without undermining the validity and integrity of the trial. The primary outcome measure is the serum level of Lp-PLA2 in the Danshen extract group and the placebo group. The secondary outcomes include the proportion of patients who show a clinically significant change, which is defined as at least a 20-point improvement in angina frequency score on the Seattle Angina Questionnaire and the carotid intima-media thickness, which will be measured using ultrasound. Other secondary efficacy and safety outcomes will also be assessed. DISCUSSION: This study will provide evidence that Danshen extract is beneficial for stable angina and may establish a possible mechanism of Danshen treatment effects on cardiovascular disease. This study may also validate an objective blood test (LP-PLA2 level) for assessing the effectiveness of Danshen therapy in patients with stable angina pectoris. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02870764 . Registered on 13 August 2016.