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    • 专利摘要:
    The present invention provides use of prostaglandin E1 (PGE1) in preparation of a medicament for intracerebral hemorrhage, and belongs to the technical field of biomedicine. Compared with the prior art, in the present invention by treating intracerebral hemorrhage with PGE1, as compared with a NaCl blank control treatment group: a patient with intracerebral hemorrhage has obviously improved prognosis functionality; a mouse with intracerebral hemorrhage has significantly improved motor ability recovery and neurological function improvement, significantly reduced number of apoptotic nerve cells and significantly increased number of viable nerve cells in perihematomal tissues, and significantly inhibited astrocyte proliferation, microglia activation and oxidative stress reactions; at the same time, in an in vitro experiment, PGE1 can reduce the decrease of cell viability, the release of LDHs and the apoptosis of neurons.
    公开号:NL2025156A
    申请号:NL2025156
    申请日:2020-03-18
    公开日:2021-04-21
    发明作者:Ke Kaifu；Shen Jiabing；Cao Maohong；Liang Jingjing
    申请人:Affiliated Hospital Of Nantong Univ；
    IPC主号:
    专利说明:

    P3447ONLOO/RR Title: USE OF PROSTAGLANDIN E1 IN PREPARATION OF MEDICAMENT FOR TREATING
    INTRACEREBRAL HEMORRHAGE USE OF PROSTAGLANDIN E1 IN PREPARATION OF MEDICAMENT FOR TREATINGINTRACEREBRAL HEMORRHAGE
    TECHNICAL FIELD The present invention provides use of prostaglandin E1 (PGE1) in preparation of a medicament for intracerebral hemorrhage, and belongs to the technical field of biomedicine.
    BACKGROUD Intracerebral hemorrhage (ICH) refers to intraparenchymal hemorrhage caused by non-traumatic spontaneous rupture of cerebral blood vessels. It is one of the subtypes of strokes and accounts for 20%-30% of all strokes in China. The ICH is a common neurological disease with acute onset and severe illness. Though many years of research, treatment methods for it are limited so far. Its acute-stage mortality rate is 30%-40%, and most survivors have sequelae such as different degrees of motor disorders, cognitive disorders, speech and swallowing disorders and so on. The high mortality and disability rates of such a disease bring heavy pain and burden to families and society. With the aging of the population, the harm caused by ICH is becoming increasingly serious. At present, the treatment of intracerebral hemorrhage is mainly medical symptomatic treatment or surgical treatment, and there is no breakthrough in other aspects, and there is no specific therapeutic drug for the protection a perihematomal tissue. In the prior art, prostaglandin E1 is mainly used clinically for myocardial infarction, thromboangiitis obliterans, arteriosclerosis obliterans and the like diseases, and there is no report on other effects of the prostaglandin E1.
    SUMMARY In view of this, an objective of the present invention is to provide use of prostaglandin E1 in preparation of a medicament for treating intracerebral hemorrhage (ICH), thereby providing a new use of the prostaglandin E1 (PGE). In order to achieve the foregoing invention objective, the present invention provides the following technical solutions. The present invention provides use of prostaglandin E1 in preparation of a medicament for treating intracerebral hemorrhage. Preferably, the prognosis of a patient with intracerebral hemorrhage is improved by protecting a perihematomal tissue after the intracerebral hemorrhage.
    -2- Preferably, the perihematomal tissue after intracerebral hemorrhage is protected by improving a local low cerebral blood flow state of the perihematomal tissue. Preferably, the perihematomal tissue after intracerebral hemorrhage is protected by promoting hematoma absorption.
    Preferably, the perihematomal tissue after intracerebral hemorrhage is protected by reducing a perihematomal injury area. Preferably, the prognosis of the patient with intracerebral hemorrhage is improved by reducing the cell viability decrease in the perihematomal tissue. Preferably, the prognosis of the patient with intracerebral hemorrhage is improved by reducing the release of a lactate dehydrogenase from the perihematomal tissue.
    Preferably, the prognosis of the patient with intracerebral hemorrhage is improved by reducing neuronal apoptosis in the perihematomal tissue.
    Preferably, the intracerebral hemorrhage is treated by conducting neuroprotection of the perihematomal tissue after the intracerebral hemorrhage.
    Preferably, the neuroprotection is achieved by one or more of inhibiting astrocyte proliferation in a perihematomal region, inhibiting microglia activation, and inhibiting oxidative stress response pathways.
    The present invention provides the use of the prostaglandin E1 in preparation of a medicament for intracerebral hemorrhage. After the prostaglandin E1 is used, the prognosis of a patient with intracerebral hemorrhage can be improved by several approaches such as protecting a perinematomal tissue after ICH, reducing a perihematomal injury area, reducing the cell viability decrease in the perihematomal tissue, reducing the release of a lactate dehydrogenase (LDH) from the perihematomal tissue, reducing neuronal apoptosis in the perihematomal tissue and conducting neuroprotection of the perihematomal tissue, etc. The protection of the perihematomal tissue after ICH is achieved by improving a local low cerebral blood flow state of the perihematomal tissue, promoting hematoma absorption and reducing the perihematomal injury area. The neuroprotection is realized by inhibiting astrocyte proliferation in a perihematomal region, inhibiting microglia activation, and inhibiting oxidative stress response pathways.
    Beneficial effects: Compared with the prior art, in the present invention by treating intracerebral hemorrhage with PGE1, as compared with a NaCl blank control treatment group: SPECT indicates that radioactive fillers at proximal and distal ends of the hematoma and perihematomal tissue are obviously improved, a rCBF value is obviously improved, and a Ra value is obviously higher than that of the control group; a mouse with intracerebral hemorrhage has significantly improved motor ability recovery and neurological function improvement, significantly reduced number of apoptotic nerve cells and significantly increased number of viable nerve cells in perihematomal
    23. tissues of the brain tissues, and significantly inhibited astrocyte proliferation, microglia activation and oxidative stress reactions in the perihematomal tissues; at the same time, in an in vitro experiment, PGE1 can reduce the decrease of cell viability, the release of LDHs and the apoptosis of neurons.
    BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chemical structural diagram of PGE1; FIG. 2 is an analysis chart of local cerebral blood flow, where A shows a PGE1 group 1 on day 5 after ICH; and B shows the PGE1 group on day 20 after ICH; C represents a control group on day 5 after ICH; and D represents a control group on day 20 after ICH; FIG. 3 shows the correlation between perihematomal hematoma volumes and Ra values; FIG. 4 shows the correlation between the volume of a perihematomal tissue and Ra values in the perihematomal region; FIG. 5 is an analysis chart of a neurological function recovery result of an ICH mice after treatment, where A shows a neurological function deficit score obtained by a 24-point method; and B shows an accelerating rotation test; N = 12, *, #p < 0.05, the difference is statistically significant compared with the control group at the corresponding time point; FIG. 6 is an analysis chart of hematoma volume and brain water content of the ICH mice, where A shows the hematoma volume and brain water content on day 4 after ICH; and B shows the hematoma volume and brain water content on day 14 after ICH; N = 6, *p < 0.05, the difference is statistically significant compared with the control group; FIG. 7 is an analysis chart of the number of degenerated neurons in the perihematomal region after ICH, where A shows the effects of ICH + NaCl, ICH + 10 pg/kg PGE1 and ICH + 20 pg/kg PGE1 on the number of FJB positive cells in the perihematomal region on day 4 and day 14 after ICH; and B shows bar statistics of A; N = 8, and 4 fields are observed for each slice, and 3 slices are taken from each brain tissue with an interval of 360 um; N = 6, *p < 0.05, the difference is statistically significant compared with the ICH + NaCl group; FIG. 8 is an analysis chart of the number of neurons in the perihematomal region, where A shows the number of neurons in the perihematomal region on day 4 after ICH after treatment with different doses of PGE1; and B shows the number of neurons in the perihematomal region on day 4 and day 14 after ICH after treatment with 20 pg/kg of PGE1; C and D are respectively bar statistics of A and B; N = 6, *, #p < 0.05, the difference is statistically significant compared with the corresponding control group; FIG. 9 shows the neurons apoptosis in the perihematomal region, where A shows the effects of different doses of PGE1 on the change in expression of activated Caspase-3 in a brain tissue in the perihematomal region on day 1, day 4 and day14 after ICH; B shows bar statistics of A; C shows the statistics of TUNEL positive cells, and D shows the TUNEL expression in the
    -4- perihematomal region on day4 and day 14 after ICH under the conditions of 10 pg/kg and 20 pg/kg of PGE1; N = 8, *, #p < 0.05, the difference is statistically significant compared with the corresponding control group; FIG. 10 is an analysis chart of astrocyte proliferation in the perihematomal region after ICH, where A shows 10 pg/kg and 20 pg/kg of PGE1 versus the astrocyte proliferation in the perihematomal region on day 4 and day 14 after ICH; and B shows bar statistics of A; N= 6, *, #p < 0.05, the difference is statistically significant compared with the corresponding control group, FIG. 11 is an analysis chart of microglia activation in the perihematomal region, where A shows
    Iba-1 expression in the perihematomal region of different treatment groups on day 4 and day 14 after ICH; and B shows bar statistics of Iba-1 positive cells in different treatment groups after ICH.
    N= 86, *p < 0.05, the difference is statistically significant compared with the corresponding control group; FIG. 12 is an analysis chart of the effect of oxidative stress injury in the perihematomal region,
    where A shows the detection of H202 content in the perihematomal tissue of different treatment groups on day 4 and day 14 after ICH; B shows the detection of MDA content in the perihematomal tissue of different treatment groups on day 4 and day 14 after ICH; C shows the detection of CuZnMn-SOD in the perihematomal tissue of different treatment groups on day 4 and day 14 after ICH; N = 6, *, #p < 0.05, the difference is statistically significant compared with the corresponding control group; FIG. 13 is an analysis diagram of cell viability of a heme-induced neuron apoptosis model (in vitro ICH model), where A shows the cell viability of primary cortical neurons under stimulation of different concentrations of hemin, as detected by CCK-8; B shows the effect of hemin on cell viability of primary neurons at different time points under the stimulation of hemin at a concentration of 50 HM; C shows the morphologies of primary cortical neurons in a bright field under a phase contrast microscope under different stimulation conditions, with a scale of 100 Hm; *p < 0.05, the difference is statistically significant compared with a Normal group; FIG. 14 is an analysis diagram of the role of PGE1 in hemin-induced neuronal injury, where A shows the cell viability of primary cortical neurons under conditions of different PGE1 concentrations, as detected by CCK-8; B shows the LDH release in the supernatant of primary cortical neurons under conditions of different PGE1 concentrations; C shows the morphologies of primary cortical neurons in a bright field under a phase contrast microscope under different stimulation conditions, with a scale of 100 um; *p < 0.05, the difference is statistically significant compared with a Hemin group;
    FIG. 15 is an analysis chart of the role of PGE1 in hemin-induced oxidative stress injury, where A shows a fluorescence staining image of TRME in cultured primary cortical neurons; B shows the fluorescence intensities of TRME under different stimulation conditions; C shows the total
    -5- ROS content in primary neurons under different conditions; p < 0.05, the difference is statistically significant compared with the Normal group. #P < 0.05, the difference is statistically significant compared with the Hemin group; and FIG. 18 is an analysis diagram of the role of PGE1 on hemin-induced neuronal apoptosis, where A shows the expression of activated Caspase-3 under conditions of different PGE1 concentrations; B shows bar statistics of A; C shows the changes in Caspase-3 activity of primary cortical neurons. D shows the changes in protein levels of Bcl-2, Bax and Cytc, E shows bar statistics of D: F shows statistics of TUNEL positive cells; G shows TUNEL changes under different PGE1 treatments. *, #p < 0.05, the difference is statistically significant compared with the control group (Hemin group).
    DESCRIPTION OF THE EMBODIMENTS The present invention provides use of prostaglandin E1 in preparation of a medicament for treating intracerebral hemorrhage (ICH). In the present invention, the prostaglandin E1 (PGE1) has a chemical name of (1R, 2R, 3R)-3-hydroxy-2(E)-(3S)-3-hydroxy-1-octenyl-5-oxo- cyclopentaneheptanoic acid, a molecular formula of C20H3405, and a chemical structural formula as shown in FIG. 1. In the present invention, the dosage form of the medicament is preferably a microsomal suspension. The present invention has no special limitation on the content of the prostaglandin E1 in the medicament, and a conventional content of the prostaglandin E1 in the medicament can be adopted.
    In the present invention, preferably ICH is treated by protecting a perihematomal tissue after the ICH. In the present invention, preferably the perihematomal tissue after the ICH is protected by one or more of improving a local low cerebral blood flow state of the perihematomal tissue, reducing the hematoma volume, and reducing the perihematomal injury area.
    In the present invention, preferably the ICH is treated by one or more of reducing the cell viability decrease, reducing the release of a lactate dehydrogenase (LDH), and reducing neuronal apoptosis. In the present invention, the cells preferably include primary cortical neurons of a C57BL/6 mouse.
    Inthe present invention, preferably the ICH is treated through neuroprotection, and in the present invention, preferably the neuroprotection effect is exerted by one or more of inhibiting astrocyte proliferation in a perihematomal tissue, inhibiting microglia activation, and inhibiting oxidative stress response pathways.
    The technical solution provided by the present invention will be described in detail in connection with the following embodiments, but they should not be construed as limiting the claimed scope of the present invention.
    Example 1
    -6- Use of PGE1 in Treatment of an ICH patient
    1. Patients: from November 2007 to January 2009, 40 ICH patients with hypertension were admitted to the Department of Neurology, Affiliated Hospital of Nantong University.
    2. Inclusion and exclusion criteria: this example was carried out according to the diagnostic criteria formulated by the National Conference on the diagnosis of Cerebrovascular Diseases in
    1995. The CT examination showed that the bleeding volume of basal ganglia was 10-30 ml. The patients were of the first onset, had no history of any chronic liver disease or hemorrhagic disease, had a platelet measurement greater than 100 x 109/L, and had a normal coagulation function. Patients with previous history of ICHs caused by encephalocoele hemarrage, subarachnoid hemorrhage and systemic diseases, and with severe functional defects of heart, liver and kidney systems shall be excluded from this example. The treatment was terminated for patients with aggravating illness, ventricular hematoma rupture or subarachnoid hemorrhage.
    3. Test grouping: this example had been approved by the Ethics Committee of the Affiliated Hospital of Nantong University, and all participants have informed consent. Patients were randomly divided into two groups according to admission dates. 40 eligible patients were divided into a PGE1 group and a control group. The PGE1 group composed of 10 males and 10 females had an average age of 50.2 + 12.2 years old and an average disease course of 10.2 + 12.2 h. The control group composed of 10 males and 10 females had an average age of 52.1 £ 5.59 years old and an average disease course of 11.8 £5.35 h.
    4. Drug therapy: all patients received routine treatment according to their conditions, including intracranial pressure reduction through dehydration, blood pressure regulation and infection control. Based on conventional therapy, the patients of the PGE1 group received intravenous injection of PGE1 at 10 pg/d for 15 consecutive days from day 5 after stroke.
    SPECT image analysis: 1) visualization: analysis was performed by two experienced nuclear medicine doctors according to a standard which stipulated that areas with reduced or enhanced radioactivity and areas with more than two layers and of a fault type were visually recognized as positive. 2) Semi-quantitative method: a region of interest (ROI) model was applied to the following layers: the largest hematoma, a central region of hematoma, a perihematomal tissue in a proximal region, a normal tissue surrounding a distal region, frontal and parietal lobes, and opposite mirror imaging regions. The uptake count of the aforementioned areas was obtained by using regular 9-pixel cyclic frames, and the uptake ratio (Ra) is defined as an obvervational index. rCBF was calculated by the semi-quantitative method. The Ra calculation formula was as follows: Ra = ROI x ray count on a lesion side/ROI x ray count on the opposite side of the mirror region. Ra value of s 0.9 or = 1.1 was of clinical significance.
    Calculation of hematoma volume and perihematomal tissue (the cranial CT showed a low density area of the perihematomal tissue): the hematoma volume was calculated by a 1/2 ABC method. The computational formula of the absolute value of the volume of the perihematomal
    -7- tissue was as follows: the absolute value of the volume of the perihematomal tissue = whole focal area showed by CT scan (mixed volume of the hematoma and the perihematomal tissue - the hematoma volume). The measurement result of the volume of the whole focal area was consistent with that of the hematoma volume. Cranial CT scan was performed on days 1, 5, 12 (day 7 after the PGE1 treatment) and 20 (day 20 after the PGE1 treatment) after the stroke.
    Clinical neurological function evaluation and prognosis evaluation: NIHSS scores were recorded on days 5, 12 and 20 after admission respectively. The mRS scores on day 1 after admission and after the treatment were recorded according to a modified Rankin scale. Evaluation of NIHSS and mRS was also conducted during the subsequent three months of follow-up.
    SPECT cerebral perfusion imaging results: Visualization: Evaluation of the SPECT cerebral perfusion imaging of the 40 patients was performed. The results showed that compared with the uninjured side, the radioactive distribution of proximal and distal ischemia in the hematoma and the perihematomal tissue was reduced. The regional cerebral blood flow (rCBF) was lower than that of the uninjured side. In the control group, the radioactive fillers at the proximal and distal ends of the hematoma and perihematomal tissue after treatment had no statistical significance. The improvement of rCBF was also not significant; however, for the treatment group, the difference of the radioactive fillers at the proximal and distal ends of the hematoma and perihematomal tissue compared with those before treatment was statistically significant, and the rCBF value was significantly increased (see FIG. 2 for the results). Semi-quantitative analysis: on day 20, the Ra values of proximal and distal ends of the perihematomal tissue in the PGE1 treatment group were significantly higher than those before treatment (p < 0.01), and significantly higher than those of the control group (p < 0.01). (See Table 1 for the results). Table 1 Changes of Ra Values of Two Groups Before and After Treatment (x £ S, n = 20) Site PGE1 treatment group (Ra value) Control group (Ra value) Day 5 Day 20 Day5 Day 20 Hematoma center 042+006 044+006 041+0.06 044+0.06 Proximal end of perihematomal tissue 0.54 £ 0.06 0.68 £0.06*# 0.53+0.08 0.52+0.06 Distal end of perihematomal tissue 0.67 10.05 0./8+0.06*%# 067x20.06 0650.06 Frontal lobe and parietal lobe regions 0.95 + 0.08 097+005 0.96+0.05 0.951+0.06 *. P< 0.01, comparison of the PGE1 group before and after treatment; and #: p < 0.01, comparison of the PGE1 group and the control group on day 20 Hematoma volume (cm3): the hematoma volumes of the PGE1 group on day 12 and day 20 were significantly reduced compared with those on day 1 and day 5, with statistically significant difference (p < 0.01). The hematoma volume of the control group was significantly reduced on
    -8- day 20 compared with that on day 1. On day 20, the hematoma volume of the PGE1 group was significantly lower than that of the control group (p < 0.01). (See Table 2 for the results). Table 2 Comparison of Hematoma Volumes of the Two Groups at Different Time Points (x £ S, cm3, n= 20) GroupsDay 1 Day 5 Day 12 Day 20 PGE1 Group 23.82+461 27.82+843 1507+7.05*A# 5401411*A# Control Group 23.85+4.53 27.9218.80 22.31+8.98 13.82+8.02* *: P< 0.01, volume comparison on day 1; A: P < 0.01, volume comparison of the PGE1 group on day 12 and day 5; # P < 0.01 comparison between the PGE1 group and the control group on day 20 Volume of perihematomal tissue: the volume of the perihematomal tissue in the PGE1 group was significantly reduced on day 20 compared with that on day 5 and that of the control group (each with p < 0.01). (See Table 3 for the results). Table 3 Comparison of perihematomal tissue volume between two groups (x + S, cm3, n = 20) Groups Day 1 Day 5 Day 12 Day 20 PGE1 Group 601+2.71 2344+820 187017.78 5.3813.13%# Control Group 5.80+2.28 23961871 20.18+7.90 12.16+6.05* * comparison on day 5, p < 0.01; # compared with the control group, P < 0.01 Neurological function scores (NIHSS score, mRS score): compared with the control group, the NIHSS score in the PGE1 treatment group had statistically significant difference on day 20 and at 3 months of follow-up (p < 0.05). For the 3 months of follow-up, the difference in mRS scores between the two groups was statistically significant (p < 0.01). (See Tables 4 and 5 for the results). Table 4 NIHSS scores of the two groups (x + S, n = 20) Groups Day 1 Day 5 Day 12 Day20 Day 20 PGE1 Group 11.85+5.99 1445+6.17 122+528 5.55+3.32* 1.50+1.00% Control Group 11.50+5.76 1405+6.02 13.35+540 9.80+421* 4051295 Comparison between the PGE1 treatment group and the control group, *P < 0.05 Table 5 mRS scores of the two groups (x £ S, n = 20) Groups Day 1 Day 20 Day 90 PGE1 Group 3.03+0.73 285+0.983 0.351049" Control Group 3.20+0.88 260+1.05 1.70+0.73 “Comparison between the PGE1 treatment group and the control group, P < 0.05 Correlation among Ra values, hematoma volumes and perihematomal tissue volume in the perihematomal region: the results showed that there was a negative correlation between
    -9- proximal and distal Ra values of the hematoma central region and perihematomal region, and volumes of the hematoma and perihematomal tissue. (See FIGs. 3 and 4 for the results).
    Adverse reactions and safety assessment of the adverse reactions: no hemorrhage, erythema and low cell proliferation was observed for the PGE1 treatment group during or after treatment. No abnormal phenomenon, such as hematuria or bloody stool, liver and kidney dysfunction, coagulation function defects or electrocardiogram abnormality, was observed.
    Example 2 Use of PGE1 in Treatment of ICH in Mice
    1. Animals: Male C57BL/6 mice of SPF grade at 26-30 g were purchased from the Laboratory Animal Center of Nantong University, and were normally fed in clean animal pegs.
    2. Experimental grouping: mice were randomly divided into a sham-operation + NaCl group, an ICH group + NaCl group, an ICH+ PGE1 (2 pg/kg) group, an ICH+ PGE1 (10 pg/kg) group, and an ICH+ PGE1 (20 pg/kg) group, totaling 5 groups with 30 mice in each group.
    3. Drug treatment: all mice were given intraperitoneal administration (normal saline or PGE1) for four times respectively at 0 h, 4 h, 12 h and 24 h after operation; and all mice received injection of approximately the same amount of liquid of about 0.5-0.6 ml.
    4. Experimental method: intracerebral injection of autologous blood was a common animal modeling method for ICH. This example was used for the therapeutic effect of PGE1 on ICH. A mouse ICH model was prepared by intracerebral injection of autologous blood, particularly including: (1) intraperitoneally anesthetizing the mice with a compound anesthetic (3 ml/Kg); (2) straightening the posture of the mouse and fixing the head of the mouse, conducting conventional skin preparation and disinfection on a local portion of the vertex cranii, and making a sagittal incision of about 1.0 cm in the middle of the vertex cranii to expose the skull; (3) locating the brain corpus striatum region at one side (by taking anterior fontanelle as the mid- point, moving forward by 0.2 mm, and opening laterally at 2.3 mm, inserting the needle at a depth of 3.5 mm) with a stereotaxic instrument, and injecting 15 kl autologous blood at a speed of 2 pl/min to prepare an operation group, and only inserting the needle without injection to prepare a sham-operation group in the same way; and (4) stitching after retaining the needle for 15 min.
    Intraperitoneal administration was carried out at 0 h, 4 h, 12 h and 24 h after the operation. NaCl was injected into the mice of the blank group, and PGE1 was injected into the mice of the PGE1 group with gradients.
    5. Neurological function score: accelerated rotation test and accelerated rotation test by a neural function deficit scoring table method: the accelerated rotation test was mainly carried out on a mouse rotator. The initial rotation speed was set at 4 r uM, the maximum rotation speed was 40 r MM, the time of the acceleration process was 8 min, and the upper limit of the movement time for each mouse was 500 s. The mice were trained 3 days before the operation, twice a day
    -10- respectively in the morning and in the afternoon (at a consistent time as much as possible); before each training, the mice moved at a speed of 4 r uM for 10 min to adapt to an unfamiliar environment followed by 2 times of accelerated rotation training, and the result of the sixth accelerated rotation was taken as a pre-operative cardinal number. After ICH operation, the accelerated rotation data of mice were tested on the afternoon of day 1, day 4, day 7, day 14 and day 28 respectively. Each mouse was measured for 3 times with a rest interval of 230 min between each time of measurement. If the time of movement exceeded 500 s, the mice were removed and the data at 500 s was recorded. Table 6 Neurological Function Deficit Scoring Table Method-24 Point Method 0 1 2 3 4 Body symmetry Normal Slight asymmetry Moderate asymmetry ~~ Apparent asymmetry Extreme asymmetry Gait (placed on a flat table) Normal Stiff, inflexible Limp, hobble Shaking, sliding Unable to walk Climbing ability (placed on a slope of 45 degrees) Normal Climbing upward at full tilt, with reduced muscle force Capable of stabilizing the body, neither sliding nor climbing Sliding down, failed to prevent the sliding down Immediately sliding down, unable to prevent the sliding down the act of turning around (placed on a flat table) None Significant one-sided turning Turning towards one side (discontinuous) Turning towards one side continuously Turning, swinging or not moving Symmetry of fore limbs (suspending the mouse by lifting the tail of the mouse) Normal Mild asymmetry Moderate asymmetry Severe asymmetry Mild asymmetry, no body/limb movement Forced turning around (placing the fore limb on a flat table, and suspending the body at the tail side by lifting the tail of the mouse) Not turning around Having a tendency of turning to one side Rotating towards one side Slowly rotating towards one side along the axis Unable to move forward/proceed The therapeutic behavior score result of the ICH mice was shown in FIG. 5. It could be seen that, the neurological function deficit score of the ICH mice treated with PGE1 was significantly lower than that of the NaCl control treatment group, and the duration of the accelerated rotation test was significantly longer than that of the NaCl control treatment group, which indicated that PGE1 could significantly improve the neurological deficit and motor function of the ICH mice and had therapeutic and protective effects. (* represented that p is smaller than 0.05).
    6. The hematoma volume after ICH was detected by H&E staining, and the brain water content was detected by a dry-wet weighing method.
    -11- The hematoma volume after ICH was measured by H&E staining. The results were as shown in FIG. 6, the hematoma volume was significantly reduced on day 14 after ICH, the hematoma volume of the control group was 6.3 + 3.73 mm3, and the hematoma volume of the group with 20 pg/kg of PGE1 was 3.7 + 2.65 mm3, such that the difference was statistically significant compared with the control group. It could be seen that 20 pg/kg of PGE1 could promote the absorption of hematoma volume on day 14 after ICH. Brain water content was measured by a dry-wet weighing method. At a specific time point after ICH, the mice were anesthetized and killed, the brain was taken, and the brain tissues on the same side of ICH, on the opposite side of ICH and of cerebellum were weighed as the wet weights, and then they were baked in a 100°C oven for 24 h and then weighed as the dry weights, and the weight of the cerebellum was used as a control. Brain water content = [(wet weight - dry weight)/wet weight x 100%]. Results were as shown in FIG. 6, the brain water content in the hemorrhagic striatum was significantly lower than that in the contralateral striatum on day 14 after ICH, while there was no significant improvement on day 4 after ICH. It could be seen that 20 pg/kg of PGE1 could relieve the brain water content in the perihematomal region after ICH.
    7. FJB staining was used for detecting the number of degenerated neurons in the perihematomal region. FJB staining was used for detecting the number of degenerated neurons in the perihematomal region, so as to understand the role of PGE1 treatment on nerve cell injury in the perihematomal region. Results were as shown in FIG. 7. FJB positive cells of the ICH + NaCl group were evenly distributed in the perihematomal region, while ICH + 10 pg/kg PGE1 reduced the number of the FJB positive cells on day 4 after ICH, but the decrease on day 14 after ICH did not reach statistical significance. The ICH + 20 pg/kg PGE1 could reduce the number of the FJB positive cells on both day 4 and day 14 after ICH, and the difference was statistically significant. The immunofluorescence technique was used for detecting the number of neurons in the perihematomal region. The loss of neurological function after ICH is closely related to the number of neurons in the perihematomal region. The immunofluorescence technique was used for detecting the number of neurons in the perihematomal region. Results were as shown in FIG. 8, the number of neurons in the perihematomal region after ICH was significantly reduced compared with that in the NaCl group. After treatment was conducted by giving different doses of PGE1, the number of neurons in the group treated with 20 pg/kg of PGE1 was increased on both day 4 and day 14 after ICH than that in the corresponding ICH + NaCl group, and the difference was statistically significant. It could be seen that 20 pg/kg could significantly reduce the neuronal apoptosis in the perihematomal region after ICH.
    -12- Western blot and TUNEL were used for detecting neuronal apoptosis in the perihematomal region. Neuronal apoptosis after ICH was an important factor of neurological deficits after ICH. The Western blot and TUNEL were used for detecting neuronal apoptosis in the perihematomal region. A brain tissue within 4 mm of the perinhematomal region was taken and homogenized, the expression of activated Caspase-3 was detected by western blot, and the results were as shown in FIG. 9. The expression of activated Caspase-3 was significantly up-regulated in the ICH + NaCl group, and when treatment with 10 ug/kg of PGE1 was given, the expression of activated Caspase-3 was reduced on day 4 after ICH compared with that of the ICH + NaCl group; and when treatment with 20 pg/kg of PGE1 was given, the expression of activated Caspase-3 was reduced on both day 4 and day 14 after ICH compared with those of the ICH + NaCl group. The TUNEL condition in the perihematomal region was further detected, and the results were as shown in FIG. 9. When 20 pg/kg of PGE1 was given, the number of TUNEL positive cells was significantly reduced compared with that of the ICH + NaCl group, and the difference was statistically significant. It could be seen that 20 ug/kg of PGE1 could significantly reduce neuronal apoptosis in the perihematomal region after ICH. An immunofluorescence technique was used for detecting astrocyte proliferation in the perihematomal region after ICH. The immunofluorescence technique was used for detecting the astrocyte proliferation in the perihematomal region after ICH. Results were as shown in FIG. 10, the number of astrocytes in the group of ICH + 20 pg/kg PGE1 was significantly reduced, which had a difference that was statistically significant compared with the ICH + NaCl group. It could be seen that 20 pg/kg of PGE1 could significantly improve the astrocyte proliferation in the perihematomal region after ICH.
    The immunofluorescence technique was used for detecting microglial activation in the perihematomal region after ICH. Microglia activation could lead to early brain injury after ICH. The immunofluorescence technique was used for detecting the expression and activation of Iba-1 in the perihematomal region so as to detect the effect of PGE1 on microglia after ICH. Results were as shown in FIG. 11, and 20 pg/kg of PGE1 could significantly reduce the number of Ibal positive cells in the perihematomal region on day 4 after ICH. It could be seen that 20 pg/kg could reduce the microglial activation in the early stage after ICH. The effects of PGE1 on oxidative stress injury in the perihematomal region after ICH were detected by indexes of H202, MDA and SOD.
    The H202 content in the perihematomal region after ICH was detected to explore the effect of PGE1 on oxidative stress injury in the perihematomal region after ICH. Results were as shown in
    -13- FIG. 12, and 20 pg/kg of PGE1 could significantly reduce the H202 level in the perihematomal region on day 4 and day 14 after ICH.
    Malondialdehyde (MDA) was a natural product of lipid oxidation in organisms.
    Lipid oxidation occurred when animal or plant cells undergone oxidative stress.
    The expression level of MDA in the perihematomal region was detected, and the results were as shown in FIG. 12. 20 pg/kg of PGE1 could significantly reduce the level of MDA in the perihematomal region on day 4 and day 14 after ICH.
    Superoxide Dismutase (SOD) was an important antioxidase in organisms, which can catalyze dismutation of anions of a superoxide to generate hydrogen peroxide (H202) and oxygen (O2). The expression level of SOD in the perihematomal region was detected, and the results were as shown in FIG. 12. The SOD content in the group treated with 20 pg/kg of PGE1 was higher than that in the NaCl group on day 4 after ICH.
    It could be seen that the treatment with 20 pg/kg of PGE1 could significantly reduce the oxidative stress level in the perinematomal region after ICH.
    Example 3 In this example, heme was used for inducing primary cortical neuron injury to construct an in vitro ICH model for the protection of nerve cells and the inhibition of oxidative stress responses.
    Establishment of a heme-induced neuronal apoptosis model (in Vitro ICH model): the mouse cerebral cortex neuronal cells from an embryo that had been cultured or 15 days was stimulated with different concentrations of hemin (Normal, DMSO, 1, 10, 50, 100 pM) for selection the best stimulating concentration.
    Results were as shown in FIG. 13. The CCK-8 results showed that with the increase of hemin concentration, the cell viability gradually decreased (the cell viability was 0.93 + 0.19 under 1 uM of hemin, the cell viability was 0.63 + 0.15 under 10 uM hemin, the cell viability was 0.45 + 0.09 under 50 uM hemin, and the cell viability was 0.25 + 0.04 under 100 MM hemin). 50 uM hemin was selected to stimulate primary cortical neurons, and the cell viability at different time points was detected.
    The cell viability was 0.67 £ 0.14 after 18 hours of hemin stimulation, and the difference was statistically significant.
    Morphologically, when hemin was 10 um, 50 uM and 100 uM, the number of neurons was significantly less than that of the Normal, DMSO and 1 uM groups, and the neurites are sparse and reduced, and the cell body disintegrated.
    In view of the above, a stimulation concentration of 50 uM hemin was selected for the subsequent study on the effectiveness of PGE1. Role of PGE1 in hemin-induced neuronal injury Different concentrations of PGE1 (10 nM, 100 nM, 1 uM and 10 uM) were selected for pre- culture 12 hours before hemin stimulation, so as to detect the effectiveness of PGE1. At 24 hours after stimulation with 50 uM of hemin, the cell viability was detected by CCK-8. The results were as shown in FIG. 14. The cell viability of the hemin-stimulated group was significantly reduced, while the cell viabilities of the groups respectively pretreated with 10 nM and 100 nM of PGE1 were significantly increased, and the increase was statistically significant compared with
    -14 - that of the hemin group.
    In order to further confirm the accuracy of this result, the LDH content in the supernatant was detected by an LDH detection kit.
    The results were as shown in FIG. 14, which were similar to that obtained by CCK-8, and both showed that cell death decreased after pretreatment with 10 nM and 100 nM of PGE1. From the morphology of neurons, after pretreated with 10 nM and 100 nM of PGE1, the neurons have intact cell bodies and slender axons compared with those of the Hemin-stimulated group.
    It could be seen that PGE1 had a protective effect on hemin-induced neuronal injury at concentrations of 10 nM and 100 nM.
    Role of PGE1 in hemin-induced Oxidative Stress Injury Iron ion Fe2+, the degradation product of Hemin, could catalyze the generation of a large number of hydroxyl radicals by participating in a Fenton reaction, thus leading to lipid peroxidation.
    It was an inducer of active oxygen.
    Mitochondrial membrane potential MMP was an important factor for maintaining the physiological function of a mitochondrial respiratory chain, and the decrease of MMP was an important indicator of cell apoptosis.
    By detecting the MMP, the oxidative stress level of cells could be understood.
    Results were as shown in FIG. 15. For the TRME fluorescence states in primary neurons under different conditions, it was observed that the TRME fluorescence intensity in the Normal group was significantly stronger than those in other groups; after Hemin stimulation, the TRME fluorescence intensity was decreased, which was only 47% of that of the control group (Normal); after pretreatment with 10 nM and 100 nM of PGE1, the MMP fluorescence intensity was significantly enhanced; and after pretreatment with 1 uM and 10 uM of PGE1, the MMP fluorescence intensity did not change significantly compared with that of the hemin-stimulated group.
    Furthermore, after the primary cortical neurons were stimulated with hemin, the intracellular total ROS content was detected by DCFH fluorescence.
    The results were as shown in FIG. 15. After hemin stimulation, intracellular ROS was increased significantly, while after pretreatment with 10 nM and 100 nM of PGE1, the ROS production was decreased compared with that of the control group, and the difference was statistically significant.
    It could be seen that pretreatment with 10 nM and 100 nM of PGE1 could reduce hemin-induced oxidative stress injury in primary cortical neurons.
    Effect of PGE1 on hemin-induced Neuronal Apoptosis The cell apoptosis under different PGE1 pretreatment conditions after Hemin stimulation was detected, so as to understand the specific effect of PGE1 on primary neuron injury.
    Results were as shown in FIG. 16, the expression level of activated Caspase-3 was significantly increased after Hemin stimulation, while the protein level of activated Caspase-3 was significantly decreased in groups respectively pretreated with 10 nM and 100 nM of PGE.
    Literature report (PMID: 26276080) showed that a Bcl-2/Bax ratio was a sensitive indicator of mitochondrial oxidative stress, and the decrease of the ratio can reflect the injury degree of cell oxidative stress.
    Through the detection of Bcl-2/Bax, the results were as shown in FIG. 16. The Bcl-2/Bax ratio was obviously reduced under the condition of Hemin stimulation, while after the
    -15- pretreatment with 10 nM and 100 nM of PGE1, the ratio was obviously increased than that of the hemin-stimulated group, and the difference was statistically significant, and the change was not obvious after pretreatment with 1 pM and 10 uM of PGE1. Furthermore, the release of a cytochrome c protein (Cytc) was further detected, and the results were as shown in FIG. 16,
    which also revealed that pretreatment with 10 nM and 100 nM of PGE1 was better than the hemin-stimulated group.
    As the gold standard for cell apoptosis, TUNEL staining results were as shown in FIG. 16. The numbers of apoptotic cells in the groups pretreated with 10 nM and 100 nM of PGE1 were significantly lower than that in the hemin-stimulated group alone.
    It can be seen that PGE 1 has a neuroprotective effect in a hemin-induced neuron model (in vitro ICH model). The above description is only preferred embodiments of the present invention.
    It should be pointed out that, for those of ordinary skills in the art, several improvements and modifications can be made without departing from the principle of the present invention.
    These improvements and modifications should also be considered as falling into the claimed scope of the present invention.
    权利要求:
    Claims (10)
    [1]
    1. Prostaglandin E1 for use in the treatment of cerebral hemorrhage.
    [2]
    Prostaglandin E1 for use according to claim 1, wherein the prognosis of a patient with a cerebral hemorrhage is improved by protecting a perihematomic tissue after the cerebral hemorrhage.
    [3]
    Prostaglandin E1 for use according to claim 2, wherein the perihematomal tissue is protected after the cerebral hemorrhage by improving a local low cerebral blood flow status of the perihematomal tissue.
    [4]
    Prostaglandin E1 for use according to claim 2, wherein the perihematomal tissue is protected after the cerebral hemorrhage by promoting hematoma absorption.
    [5]
    Prostaglandin E1 for use according to claim 2, wherein the perihematomal tissue is protected after the cerebral hemorrhage by reducing a perihematomic lesion area.
    [6]
    Prostaglandin E1 for use according to claim 1, wherein the prognosis of the patient with a cerebral hemorrhage is improved by decreasing the cell viability in the perihematomal tissue.
    [7]
    Prostaglandin E1 for use according to claim 1, wherein the prognosis of the patient with a cerebral hemorrhage is improved by decreasing the release of a lactate dehydrogenase from the perihematomal tissue.
    [8]
    The prostaglandin E1 for use according to claim 1, wherein the prognosis of the patient with a cerebral hemorrhage is improved by decreasing neuronal apoptosis in the perinematomal tissue.
    [9]
    Prostaglandin E1 for use according to claim 1, wherein the cerebral hemorrhage is treated by performing neuroprotection of the perihematomal tissue after the cerebral hemorrhage.
    [10]
    Prostaglandin E1 for use according to claim 9, wherein the neuroprotection is achieved by one or more of inhibiting astrocyte proliferation in a
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    CN110507659A|2019-11-29|
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    CN201910822127.4A|CN110507659A|2019-09-02|2019-09-02|Application of the prostaglandin E1 in the drug of preparation treatment cerebral hemorrhage|
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