| Cerebrovascular disease is becoming a prominent public health concern.Due to high rates of morbidity, disability and mortality, this type of diseasenot only has a strong impact on the quality of life, but also accompanied byheavy economic burdens for the patients’ families and society in general.Currently, treatment of this disease is accomplished by surgically removingblocked blood vessels in order to improve the blood supply in and around thelesion. Once the blood supply improves, this area is extremely prone to getsevere cerebral reperfusion injury. There are several pathological mechanismsin the process of cerebral ischemic such as excessive release of excitatoryamino acids, loss of ionic homeostasis, energy failure, inflammatory response,increased oxidative stress and apoptosis. These mechanisms eventually causeirreversible damage of the brain tissue. So recent years, chemical drugs suchas calcium ion antagonist and radical scavengers as well as neuroprotectiveagents have been used for the treatment of CIR injury. However, side effectssuch as resistance to drugs, cerebral hemorrhage and gastrointestinalirritationmay exceed the clinical benefits for long-term therapy. Fortunately,clinical applications and experimental reports of traditional Chinese medicinesagainst CIR injury have been ascendant.Radixet rhizoma Rhei, dried roots and rhizome of Rheum officinale Baill,Rheum palmatum L, and Rheum tanguticum Maxim has been used as animportant traditional Chinese medicine in Chinese folk for a long history,which widely grows in Sichuan, Qinghai, and Gansu and so on. Sheng Nong’sherbal classic\said that it was “bitter in taste, cold-natured, into the spleen,stomach, large intestine, and pericardium, liver” and had the effects ofanti-indigestion, anti-fever, discharging fire, cooling blood, removing stasisand detoxicate. Modern pharmacological studies show that it could be used for purging heat flux of intestinal, removing pattogenic heat from the blood andtoxic material from the body, decreasing stagnated. Besides, it could have theeffects of anti-radical, anti-hyperlipidemia, anti-arteriosclerosis, and anti-cancer, anti-aging. The main medicinal composition of Rhubarb is anthraqui-none compound, which has the utility monomer Chry that belonging toanthraquinone compound. Chry have ability of anti-cancer, anti-bacterial,anti-inflammatory, depressurization, and spasmolysis.Chry can scavenge O2-, DPPH free radical in vitro, and inhibit lipidperoxidation in liver and brain tissue of rat. Chry has also been shown toimprove learning and memory function and to improve tolerance ability ofhypoxia CIR injury in mice. However, because of the lack of researchevidence in level of molecule, whether Chry protects against CIR by blockingapoptosis pathway remains unknown.However, Chry is indissolvable in water, irritates the stomach, has lowbioavailability, and its physical and chemical properties are unstable. Thesedisadvantages restrict applications of Chry in the clinical setting. In this study,we extracted crude product of Chry from Rhubarb, then separated monomerby PHPLC, finally completed the preparation of Chr-lip; kunming mice wereinduced into CIR by transient middle cerebral artery occlusion, neurologicaldeficits, neuronal ultrastructure, histopathological changes, the activity ofSOD, GSH-PX, NOS, the content of MDA, NO, expressions of Bax, Bcl-2,Cytc, caspase3were examined dynamic observed, then the relation wereconfirmed between oxidative damage, apoptosis with cerebral ischemia.Furthermore, Male mice were intraperitoneally injected with Chr-lip for threesuccessive days, and then subjected to brain ischemia induced by MCAO.After reperfusion24h, neurological deficits, neuronal ultrastructure,histopathological changes, oxidative stress-related biochemical parameters,neuronal apoptosis, and apoptosis-related proteins were assessed. The presentstudy was designed to evaluate the neuroprotective effects of Chr-lip, as wellas the underlying mechanisms by focusing on oxidative stress and neuronalapoptosis. The study was divided into three part list as below. PartⅠ Extraction of Chrysophanol and preparation of ChrysophanolliposomesObjective: PHPLC method was established for purifying the substancesof Chry from Rhubarb, the prescription and preparation technology of Chr-lipwas investigated; the quality of Chr-lip was evaluated.Methods: HPLC method was developed for determineation of Chry. Thecolumn was a Hypersil BDS C8(4.6×150mm,5μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, ata flow rate of1.0mL·min-1. The column temperature was at35℃. Thedetection wavelength was254nm. Second, PHPLC method was establishedfor purifying the substances of Chry. The column was ZORBAX SB-C18:(21.2mm×250mm,7μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, at a flow rate of20mL·min-1. The column temperature was at35℃. The detection wavelengthwas254nm. Fraction was collected: based on the peak, the threshold beingMin:2.2. Both NMR and HPLC are used for the structure and quantitativeanalysis of Chry. The prescription and preparation technology of Chr-lip wasinvestigated; film-ultrasonic dispersion method was used to prepare Chr-lip;the encapsulation efficiency, morphology, size distribution and stability qualityof Chr-lip was evaluated.Results: NMR analysis showed that the structure was Chry. HPLCanalysis showed that the purity of Chry was98.9%, the entrapment efficiencyof Chr-lip was88.5%. The grain diameter was relatively homogeneous, therewas no gathered phenomenon, and the grain diameter was less than2microns.Conclusions: PHPLC method was high sensitivity, easy operated forpurifying the substances of Chry. The purity of Chry was98.9%; theentrapment efficiency of Chr-lip was88.5%. It can be used for pharmacolo-gical study.PartⅡ Effects of Chrysophanol liposome on cereral ischemia-reperfu-sion injury induced oxidative stress in miceObjective: Neurological deficits, neuronal ultrastructure, histopatholo- gical changes, the activity of SOD, GSH-PX, NOS, the content of MDA, NO,were dynamic observed, then the relation were confirmed between oxidativedamage with CIR injury. We estimated the anti-oxidative of Chr-lip on CIRinjury in mice and explore its possible mechanisms.Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chi-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neurological deficits of each mousewere determined; changes of neuronal ultrastructure, histopathological weremeasured by transmission electron microscopy, HE stainning; then the activityof SOD, GSH-PX, NOS, the content of MDA, NO were determined.Results:1Chr-lip alleviated neurological deficit: in the control and sham group,there were no obvious neurological functions in mice. In model groups theneurological scores remarkably increased comparing with sham group(P<0.01), the highest neurological scores of group was at reperfusion24h.The results showed that neurological deficit was induced by CIR in mice.After CIR24h, neurological deficit score of the model group wassignificantly higher than that of the sham group (P<0.01); whereas those ofChr-lip-treated groups (10.0,5.0,0.5mg·kg-1) were significantly lower thanthe value in the model group (P<0.05~P<0.01).2Chr-lip improved neuronal ultrastructure injury: in the control and shamgroup, neuron structure was regular, abundant organelles could be seen. Afterischemia reperfusion3h, shrunken neucleus and aggregated chromatin toward the nuclea membrane were observed. Obviously, slightly swollen mitocho-ndrion and vacuolus were observed. After ischemia reperfusion6h, neuronnuclear chromatin density increased, the nuclear membrane have seriousshrinkage, side set, partly dissolved mitochondria, endoplasmic reticulumpartly dissolved, cytoplasm height edema, vacuoles were observed, thenumbers of organelles reduced. After ischemia reperfusion12h, karyopy-knosis, swollen nucleus, edge chromatin, a large number of vacuolation incytoplasm were observed, mitochondria had completely dissolved. Afterischemia reperfusion24h, organelles were significantly decreased, vacuolatedmitochondria structure, disappeared outer, apoptosis body, and roughendoplasmic reticulum degranulation were observed. After ischemia reperfu-sion48h, karyopyknosis, nuclear membrane invagination, chromatin clumpsblock edge set under nuclear membrane, height edema cytoplasm, less numberof organelles, different degree of empty mitochondria, and lysosome wereseen. After ischemia reperfusion72h, karyopyknosis, nuclear membranedissolved, chromatin clumps block edge set under nuclear membrane, heightedema cytoplasm, different level of mitochondrial breakage, rough endoplas-mic reticulum degranulation, less number of organelles were seen. The resultsshowed neuronal ultrastructure was changed by CIR in mice. In the treatmentgroups, the chromosome distribution was relatively uniform, and the nuclearmembrane was clear. The mitochondria were reduced to some extent, butswelling of the rough endoplasmic reticulum was mild.3Chr-lip alleviated histopathological changes: the histopathologicalexamination revealed that there was no obvious damage in the control andsham group. After ischemia reperfusion3h, mild edema in infarcts ofsurrounding brain tissue, mott cell in intercellular space with a small amountof inflammatory cell infiltration, pyknosis degeneration of neurons and glialcells were seen; there was no clear cell necrosis; with the reperfusion6h-12h,the above change gradually aggravated; with the reperfusion24h-48h,karyopyknosis, thick dyeing, condensed chromatin gathered around thenucleus with the change before the apoptosis or apoptosis, significant damage of neuron structure were seen; after ischemia reperfusion72h, cell edemagradually alleviated, edema surrounding part of the nerve cells, inflammatorycell infiltration gradually reduced, gliocyte proliferation were observed. Theresults showed histopathological were changed by CIR in mice. Chr-lipmarkedly alleviated histopathological changes induced by CIR.4Chr-lip enhanceds antioxidant ability: the content of MDA reached apeak by12h, and was maintaining from24to48hours. Compared with shamgroup, the content of MDA significantly increased at different time points ofreperfusion (P<0.01). The activity of SOD reached a nadir by12h, andincreased from24to72hours. Compared with sham group, the activity ofSOD significantly decreased at different time points of reperfusion (P<0.01).The activity of GSH-PX reached a nadir by24h, and increased from48to72hours. Compared with sham group, the activity of GSH-PX significantlydecreased at different time points of reperfusion (P<0.01). The content of NOreached a peak by6h, and maintained from12to24hours. Compared withsham group, the content of NO significantly increased at different time pointsof reperfusion (P<0.01). The activity of NOS reached a peak by12h, andincreased from48to72hours. Compared with sham group, the activity ofNOS significantly decreased at different time points of reperfusion (P<0.01).The results showed that the antioxidant ability was decreased induced by CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the content of MDA (P<0.05~P<0.01), NO (P<0.05~P<0.01), the activity of NOS (P<0.01, P<0.05, P＞0.05), enhanced theactivity of SOD (P<0.01), GSH-PX (P<0.05~P<0.01).Conclusion: Neurological deficits, neuronal ultrastructure injury,pathologic histology injury, the activity of SOD, GSH-PX, NOS, the contentof MDA, NO, were dynamic changed, as well as cerebral ischemia reperfusion12to24hours was an important time turning point in the process of cerebralischemic injury. We demonstrated that Chr-lip protected against CIR injury byimproving neurological, neuronal ultrastructure and histological deficits, andthese beneficial effects were associated with inhibition of oxidative stress, such as elevation of SOD and GSH-PX activities, reduction of the activity ofNOS, the content of MDA, NO. So the protective mechanisms of Chr-lipagainst CIR injury might be involved to its anti-oxidant activities.PartⅢ Effects of Chrysophanol liposome on cereral ischemia-reperfus-ion injury induced apoptosis in miceObjective: neuronal apoptosis, the expression of Bax, Bcl-2, Cytc,caspase3, were dynamic observed, and then the relation was confirmedbetween oxidative damage with CIR injury. We estimated the apoptosis ofChr-lip on CIR injury in mice and explore its possible mechanisms.Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chr-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neuronal apoptosis were detected byHoechst33258stainning; Immunohistochemistry was used for measuring thepositive cells of Bax, Bcl-2, Cytc, caspase3. The protein and mRNAexpression of Bax, Bcl-2, Cytc and caspase3were detected by western blotand real-time quantitative PCR.Results:1Chr-lip attenuated the neuronal apoptosis: neuronal injuries in theischemic hemispheres were analyzed by Hoechst33258staining. Apoptoticcells were sparsely detected in the control and sham group; after ischemiareperfusion3h, there was a small amount apoptotic cell with chromosomehyperchromatic and pyknosis; with the reperfusion extend, the number ofapoptotic cell with shrink and chromatin agglutination increased; however, after ischemia reperfusion48h, the number of apoptotic cell with nuclearhyperchromatism, pyknosis decreased. Compared with sham group, there wasa significant difference in the number of neurons apoptosis between modelgroups (P<0.05). Neuronal injuries in the ischemic hemispheres were analyzedby Hoechst33258staining. The treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)effectively attenuated the neuronal apoptosis caused by CIR injury, asindicated by significant reduction of apoptotic rate (P<0.05~P<0.01).2Chr-lip effected on the positive cell of apoptosis-related: the number ofBax, Cytc, caspase3positive cell increased gradually, reached a peak atreperfusion24h, decreased from reperfusion48to72hours; compared withsham group, the number of Bax, Cytc, caspase3positive cell obviouslyincreased at different time points of reperfusion (P<0.01). The number ofBcl-2positive cell decreased gradually from reperfusion3to24hours, thenumber of Bcl-2positive cell reached a nadir at reperfusion24h, increasedfrom reperfusion48to72hours; compared with sham group, the number ofBcl-2positive cell obviously decreased at different time points of reperfusion(P<0.01). The results showed that the number of Bax, Cytc, and caspase3positive cell increased, Bcl-2positive cell obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the number of Bax positive cell, Cytc positive cell,caspase3positive cell (P<0.05~P<0.01), increased the number of Bcl-2positive cell (P<0.05~P<0.01).3Chr-lip effected on the proteins of apoptosis-related: the proteins ofBax, Cytc, caspase3increased gradually, reached a peak at reperfusion24h,decreased from reperfusion48to72hours; compared with sham group, theprotein of Bax, Cytc, caspase3levels was obviously up-regulated (P<0.01);the protein of Bcl-2decreased gradually, reached a nadir at reperfusion24h,increased from reperfusion48to72hours; the protein of Bcl-2levels wasobviously down-regulated (P<0.05~P<0.01). The results showed that theproteins of Bax, Cytc, and caspase3increased, Bcl-2obviously decreased afterCIR. Compared with model group, treatment with Chr-lip (10.0,5.0mg·kg-1) significantly reduced the protein of Bax, Cytc, and caspase3(P<0.01),increased the protein of Bcl-2(P<0.01). Treatment with Chr-lip (0.5mg·kg-1)significantly reduced the protein of Bax, Bcl-2, Cytc, caspase3showedsignificant differences (P<0.05~P<0.01).4Chr-lip effected on the mRNA expression of apoptosis-related:compared with sham group, the mRNA levels of Bax, Cytc, caspase3wasobviously up-regulated (P<0.05~P<0.01), reached a peak at reperfusion24h;the mRNA levels of Bcl-2was obviously down-regulated (P<0.05~P<0.01),reached a nadir at reperfusion24h. The results showed that the mRNA levelsof Bax, Cytc, and caspase3increased, Bcl-2obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the mRNA expression of Bax, Cytc, and caspase3(P<0.05~P<0.01), increased the mRNA expression of Bcl-2(P<0.01).Conclusion: Neuronal apoptosis, the expression of Bax, Bcl-2, Cytc andcaspase3, were dynamic changed, as well as cerebral ischemia reperfusion24h was an important time turning point in the process of cerebral ischemicinjury. We demonstrated that Chr-lip protected against CIR injury by reducingneuronal apoptosis, promoting of Bcl-2expression, inhibiting of Baxexpression and Cytc release, and suppressing of caspase3activation. So theprotective mechanisms of Chr-lip against CIR injury might be involved to itsanti-apoptotic. |
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