Protective Effect Of THSG On Cardiotoxicity Induced By Doxorubicin In Vitro And In Vivo

Part 1Protective effect of THSG on cardiotoxicity induced bydoxorubicin in vitro and in vivoDoxorubicin (also named adriamycin) is an anthracycline antibiotic that has beenused for more than 30 years for the treatment of a wide variety of cancers. However, severecardiomyopathy and heart failure have been observed in DOX-treated cancer patient, whichlimits the clinical chemotheraphy. It has been proposed that doxorubicin-inducedcardiomyopathy is at least partially caused by increased oxidant production in the heart, andthere is a great deal of supportive evidence for this hypothesis.Tetrahydroxystilbene-glucoside(THSG) was the antioxidative component of Polygonummultiflorum Thunb and it showed strong antioxidant activity in vitro. Previous researcheselucidated THSG could diminish peroxidation level of brain in Alzheimer's Disease modelmice and ischemia-reperfusion model mice. Structurally, THSG belongs to hydroxystilbene,and its structure is similar to that of resveratrol extracted from red wine which hassignificantly protective effects on cardiomyocyte injury. Based on the previous reportsthat THSG protects normal tissues against oxidative stress, we envisioned a possibility thatTHSG might protect heart from DOX injury.In the present study we used neonate rattus cardiomyocytes in vitro and acutemouse model of DOX-induced cardiotoxicity to examine the protective effect of THSG.§1. Protective effects and mechamism of THSG on DOX induced actue cardiotoxicityin mice1. Protective effects of THSG on DOX-induced cardiotoxicity in mice Method: A single DOX (15 mg/kg) was intraperitoneally injected to the animals to inducedthe actue cardiotoxicity, THSG (0.1g/kg·d) were administered with intragastric injection(p.o.) for 7 days continuously. Then it was observed whether THSG have protective effectson DOX-induced cardiotoxity in mice.Results:â‘ Animal body weight and survival: There was no difference on body weightamong 3 groups at day 1. On the 7th day after DOX administration, animals' body weightswere reduced(21.8±3.6 g) by DOX treatment compared with control(25.6±2.6 g,P＜0.01). Treatment with THSG did not increase body weights of animals (22.5±2.8 g,P＞0.05 compared with DOX alone). There was no difference in the heart weight/bodyweight ratio among the 3 groups(4.5±0.6mg/g in control group, 4.3±0.7mg/g in DOXgroup and 4.3±0.4 mg/g in THSG group). The survival rate of animals in the control groupwas 100%. DOX treated animals had a compromised survival rate (70%), and there was atrend of increased survival in THSG treated animals (85%) compared with DOX treatedgroup (P＞0.05, log rank test).â‘¡Histological findings : Normal heart histologicalfindings were seen in the control group.On the other hand, there were histological changesin DOX group by the presence of marked interstitial oedema, focal subendocardial bleed,cardiac muscle fibre destruction or loss, which was qualitatively recognized as DOX-inducecardiac damage. The lesions were significantly reduced in the groups treated with THSGcompared to DOX group.2. Effects of THSG on serum LDH and CK in DOX administrated miceMethod: Serum LDH and CK were investigated with biochemical method.Results: On the 7th day after DOX adminstration, serum LDH increased from 3.87±0.40U/ml of normal control group to 6.00±0.27 U/ml of the DOX group (P＜0.01). However,THSG (0.1g/kg) could prevent the increase of serum LDH induced by DOX, with the value4.86±0.20U/ml (P＜0.05, compared with DOX alone). Serum CK increased from 1.55±0.49 U/ml of control group to 3.02±0.28U/ml of the DOX alone administrationgroup (P＜0.01). THSG administration could inhibit the increase of serum CK induced byDOX, with the value of 1.94±0.4U/ml (P＜0.05, compared with DOX alone)。3. Effects of THSG on MDA and GSH in DOX administrated miceMethod: MDA and GSH were investigated with biochemical method.Results: On the 7th day after DOX adminstration, MDA content increased from 1.15±0.30nmol/mg protein of normal control group to 4.17±0.91 nmol/mg protein of the DOX group(P＜0.01). However, THSG (0.1g/kg) could significantly prevent the increase of MDAinduced by DOX, with the value 2.32±0.76 nmol/mg protein (P＜0.01, compared withDOX alone). GSH content decreased from 24.43±6.57mg/g protein of control group to13.65±3.46 mg/g protein of the DOX alone administration group(P＜0.01). THSGadministration could inhibit the decrease of GSH induced by DOX, with the value of18.31±3.27 mg/g protein (P＜0.01, compared with DOX alone).§2 Protective effects of THSG on DOX induced cardiomyocyte lesion1. Effects of THSG on viability of DOX treated cellsMethod: Cell viability was determinated by MTT assay.Results: Cell viability of control group was 100%. DOX markedly affected cell survival asthe rate of cell survival after exposure to 1μM DOX for 24h was 72.68±8.52%(P＜0.01),while in the presence of THSG(3,10,30,100,300μM), cell viability was significantlyenhanced in a concentration-dependent manner with the values of 76.09±10.24 %, 81.37±9.84 %, 83.43±11.47 %, 91.21±7.54 %, 95.24±10.87 % respectively. Except 3μMTHSG group, 10-300μM THSG groups had significant difference compared with the DOXalone group (P＜0.05, P＜0.01). 2. Effects of THSG on DOX-induced apoptosisMethod: Cell apoptosis was investigated by TUNEL assay.Results: The rate of apoptotic cells of control group was 4.57±0.52%. DOX markedlyincreased the rate of positive cells after exposure to 1μM DOX for 24h with the value was32.55±3.47% (P＜0.01), while in the presence of THSG(10,30,100,300μM), the rate ofapoptotic cells was significantly decreased in a concentration-dependent manner with thevalues 26.53±3.27%,20.12±2.64 %,13.34±8.64 %,10.48±1.57 % respectively. Allgroups had significant difference compared with the DOX alone group (P＜0.05, P＜0.01).3. Effects of THSG on DOX-induced loss of mitochondrial membrane potentialMethod: Mitochondrial membrane potential was investigated by fluorescence probe.Results: Nearly all cells were well spread and exhibited red or orange fluorescence in anuntreated culture, Quantitative analysis showed ratio of JC-1 aggregates/monomericwas14.52±3.86 %. In contrast, a culture treated with DOX for 24h demonstrated multiplerounded cells, a majority of which fluoresce green exclusively with the value of 0.61±0.16% (P＜0.01, compared with control), indicating loss of mitochondrial membrane potential.A culture treated with DOX in the presence of THSG(10,30,100,300μM) appeared similarto untreated control, with spread cells exhibiting red or orange fluorescence with thevaluesl.48±0.34%, 2.32±0.46 %,3.70±0.87%,5.35±1.25% respectively. Except 10μMTHSG group, 30-300μM THSG groups had significant difference compared with DOXgroup (P＜0.01).4. Effects of THSG on DOX-mediated ROS generationMethod: ROS were investigated by fluorescence probe.Results: Quantitative results (green fluorescence area) are expressed as % of values foundin cells untreated. The value was increased from 100% of control group to 337.56±45.56 % after exposure of 1μM DOX for 24h (P＜0.01), indicated that DOX stimulatedsignificant increase of ROS level. A culture treated with DOX in the presence of THSG(30,100,300μM) could inhibited DOX-mediated ROS generation with the values 265.65±23.46 %, 227.70±26.57%, 184.17±34.52%. All groups had significant differencecompared with the DOX alone group (P＜0.05, P＜0.01).5. Effects of THSG on apoptosis-related proteins expressionMethod: Protein expressions were investigated by Western blot.Results:â‘ Treatment of the cells with 1μM DOX for 24h induced the cleavage ofprocaspase-3(32 kDa) to its 17 kDa subunits. Caspase-3(17 kDa)/β-actin density ratio wassignificantly increased from normal control group 0.08±0.01 to DOX group 1.31±0.27(P＜0.01). THSG (30,100,300μM) could inhibit caspase-3 protein activation indose-dependent manner, and density ratio were 0.80±0.14,0.42±0.06,0.12±0.13. Allthese THSG groups had significant difference from DOX alone group (P＜0.05, P＜0.01) .â‘¡DOX treated for 24h markly increased Bcl-2 protein expression. Bcl-2/β-actin densityratio was significantly increased from normal control group 0.42±0.04 to DOX group0.88±0.09 (P＜0.05). THSG (30,100,300μM) could dose-dependently make this potentiallyadaptive response more outstanding, and density ratio were 0.99±0.10, 1.37±0.08,1.64±0.12. Except 30μM THSG group, 100 and 300μM THSG groups had significantdifference from DOX alone group (P＜0.05, P＜0.01).â‘¢DOX treated for 24h marklyincreased Bax protein expression. Bax/β-actin density ratio was increased from normalcontrol group 0.64±0.07 to DOX group 1.09±0.12 (P＜0.05). THSG (30,100,300μM) coulddose-dependently inhibit the increase of Bax protein expression, and density ratio were0.95±0.08, 0.75±0.06, 0.66±0.05. Except 30μM THSG group, 100 and 300μM THSGgroups had significant difference from DOX alone group (P＜0.05, P＜0.01). 6. Effects of THSG on DOX-induced increase of [Ca²⁺]_iMethod: [Ca²⁺]_i was investigated by fluorescence probe.Results: Treatment of cardiomyocytes with DOX greatly increased level of [Ca²⁺]_i. TheCa²⁺ increase was very quick and did not reach plateau at 5 min. Quantitative analysisshowed fluorescence intensity after treatment of DOX /basal value was 2.33±0.43.Pretreatment of THSG inhibited DOX-induced increase of [Ca²⁺]_i. It reached plateau atapproximately 2 min with pretreatment of 30μM THSG and the ratio was 1.33±0.07.Treatment of 100 and 300μM THSG nearly maintained basal [Ca²⁺]_i after DOX was addedwith the value 1.09±0.04 and 1.06±0.02. All these THSG groups had siginificant differencefrom DOX group (P＜0.01).Conclusions:1. THSG had protective effects on acute cardiotoxicity induced by DOX in mice, and itseffects may be associated with inhibiting oxidative damage by DOX.2. THSG could inhibit DOX-induced apoptosis and it may be involved that it preventedDOX-mediated loss of mitochondrial membrane potential, caspase-3 activation andchanges of Bax and Bcl-2 protein expression.3. THSG could inhibit DOX-mediated ROS generation.4. THSG could inhibit the increase of [Ca²⁺]_i induced by DOX. Part 2The electrophysiological mechanism of calculus bovissativus on analgesic effectCalculus bovis has been used for thousand years as the Chinese traditional medicine.The classical Chinese patent medicine NiuHuang JieDu Wan and NiuHuang QianJing Shanwhose major ingredient were calculus bovis had antipyretic and analgesic effect andapproved for clinical use. Previous reports had verified that calculus bovis and culturalcalculus bovis could relieve pain induced by acid in mice. We presumed that themechanism of calculus bovis on analgesic effect was not only involved itsanti-inflammatory effect but also the directly intervention of afferent pain sense. In order toresolve the shortage raw material of calulus bovis, the national management of drugsurveilance had validated in succession three substitutes since 1972: artificial bezoar,cultural calulus boris and calulus bovis stativus (CBS).CBS is prepared in ox bile bysimulating the biochemical process of bilirubin calcium formation and by employingbioengineering techniques. It has same quality as that of natural calculus bovis in terms ofproperties, structure, component and content.In the present study we used sciatic nerve trunk of toads and cultured adult rattrigeminal ganglion neurons to examine the effect of CBS on action potential, currents ofvoltage-dependent sodium channel and voltage-dependent calcium channel.1. The effect of CBS on action potential of sciatic nerve trunk of toadsMethod: The action potential of sciatic nerve trunk of toads was recorded by RM6240BDpolygraph.Results: 0.4 mg·mL^-1 CBS could solfly decrease the action potential of satatic nerve trunkof toads. The amplitude of action potential was decreased from 2.68±0.26 mV to 2.55±0.29 mV of CBS treatment for 30min. The inhibitory rate was 5.0±2.4% (n=7, P＞0.05). 4mg·mL^-1 CBS could significantly decrease the action potential of satatic nerve trunk oftoads in time-dependent manner. The inhibitory rates were 21.3±10.3%, 36.8±14.2%,42.5±17.6% respectively when CBS treated for 10, 20 and 30min (n=8, P＜0.01), washed byRinger solution for 30 min could recovery the action potential partly. Under the samecondition, treated satatic nerve trunk of toads with 0.02% lidocaine for10, 20 and 30min,the inhibitory rates of action potential were 26.8±14.9 %, 48.0±19.1%, 60.7±14.2 %respectively (n=6, P＜0.05). Dissolvant control had no effect on action potential.2. The effect of CBS on total currents of voltage-dependent sodium channel (I_Na-T).Method: whole cell patch clamp record.Results: The amplitude of total currents before treatment was 12.1±0.5 nA and the valuewas 12.1±0.6 nA (n=5, P＞0.05) after 0.05% dissolvent treatment, indicated that thedissolvent had no effect on I_Na-T. CBS could dose-dependently inhibit the total currents ofvoltage-dependent sodium channel on trigeminal ganglion neurons (TRG). The inhibitoryrates of 0.2, 2 and 20μg·mL^-1 CBS on I_Na-T were 25.9±5.7%, 45.0±7.6%, 55.8±7.8%respectively (n=6, P＜0.01). Treatment with 20μg·mL^-1 CBS decreased the currents from12.2±0.7 nA to 5.4±0.9 nA and washing could not make it recovery(5.6±1.0 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_Na-T under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL^-1 CBS treatment (control: V_1/2=-19.0±10.6 mV,k=4.3±1.7; 20μg·mL^-1 CBS:V_1/2=-24.5±10.3 mV, k=4.9±2.5 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_Na-T. 3. The effect of CBS on TTX-resistant currents of voltage-dependent sodium channel(I_Na-TTX-r).Method: whole cell patch clamp record.Results: The dissolvent had no effect on I_Na-TTX-r. CBS could dose-dependently inhibitthe TTX-resistant currents of voltage-dependent sodium channel on TRG. The inhibitoryrate of 0.2, 2 and 20μg·mL^-1 CBS on I_Na-TTX-r were 8.1±2.0%, 28±5.2%, 39.3±5.7%respectively(n=6, P＜0.01). Treatment with 20μg·mL^-1 CBS decreased the currents from11.4±1.4 nA to 6.9±0.6 nA and washing could not make it recovery(6.8±0.8 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_Na-TTX-r under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL^-1 CBS treatment.( control: V_1/2= -24.1±0.2 mV,k=1.6±0.1; 20μg·mL^-1 CBS: V_1/2= -23.0±1.4 mV, k=1.6±0.2 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_Na-TTX-r.4. The effect of CBS on the currents of voltage-dependent calcium channel (I_Ca).Method: whole cell patch clamp record.Results: The amplitude of the currents before treatment was 5.3±0.8 nA and the value was5.1±0.6 nA (n=6, P＞0.05) after treated with 0.05% dissolvent, indicated that the dissolventhad no effect on I_Ca. CBS could dose-dependently inhibit the currents of voltage-dependentcalcium channel on TRG. The inhibitory rate of 0.2, 2, 20μg·mL^-1 CBS on I_Ca were30.3±4.7%, 41.9±3.6% and 56.7±6.8% respectively (n=6, P＜0.01). Treatment with 20μg·mL^-1 CBS decreased the currents from 5.7±0.9 nA to 2.5±0.4 nA and washing couldnot make it recovery(2.4±0.6 nA, P＞0.05). Current-voltage (I-V) relation of I_Ca underdifferent concentration showed CBS had no effect on maximum activation potential andthreshold of activation potential. Conductance-voltage (G-V) relation showed no significantchange after 20μg·mL^-1 CBS treatment( control:V_1/2=-15.5±3.2 mV, k=3.4±0.8; 20 μg·mL^-1 CBS:V_1/2= -12.3±4.6 mV, k=4.5±0.6 (n=6, P＞0.05)), indicated that CBS had noeffect on activation kinetics of I_Ca. Steady-state inactivation curves showed no significantchange after 20μg·mL^-1 CBS treatment( control:V_1/2= -36.3±4.5 mV, k=21.4±5.4; 20μg·mL^-1 CBS: V_1/2= -33.5±5.8 mV, k=18.5±4.3 (n=6, P＞0.05)), indicated that CBS hadno effect on inactivation kinetics of I_Ca.Conclusion:The inhibitory effect of CBS on the voltage-dependent sodium and calcium currents maycontribute to its relieving pain.