CN106176694B - A kind of over-saturation methane physiological saline and its preparation method and application - Google Patents
A kind of over-saturation methane physiological saline and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to field of medicaments, more particularly to a kind of over-saturation methane physiological saline and its preparation method and application.Sterile saline and methane gas according to volume ratio are 3 by a kind of preparation method of over-saturation methane physiological saline：0.8-1.2 input internal layers are covered in the aluminium foil bag of polyethylene film, until pressure reaches 0.18-0.22MPa in aluminium foil bag；Aluminium foil bag is handled 6-8 hours under the pressure of 0.45-0.55MPa；Then methane gas is inputted in aluminium foil bag with complement lysis in the part of physiological saline, and the pressure in aluminium foil bag is made to be maintained at 0.18-0.22MPa.This method makes methane gas be dissolved in the level for reaching in physiological saline and tending to over-saturation or saturation, and methane gas content is high, prepares easy.The present invention also provides over-saturation methane physiological saline to prepare the application in treating Nrf2 signal path drugs, the especially application in Reperfusion Injury of Spinal Cord drug.
Description
Technical field
The present invention relates to field of medicaments, in particular to a kind of over-saturation methane physiological saline and preparation method thereof and
Using.
Background technology
Spinal cord injury is still considered as being a kind of the sick and wounded of no special therapeutic method so far.People damage spinal cord in the past 20 years
The cause of disease and mechanism of wound have carried out a large amount of basic research and clinical observation, it is understood that the secondary damage after Primary damage
Evil, if ischemical reperfusion injury is a key factor for causing neurotrosis.
Reperfusion Injury of Spinal Cord is to be difficult to expect complication after thoracoabdominal aortic aneurysm and spinal surgery, is occurred
Rate is up to about 13-18%, damages the quality of life for causing paraplegia or quadriplegia to seriously affect patient.However, up to the present
Still without effectively preventing measure.Studies have shown that after ischemia of spinal cord in refilling process, a large amount of active oxygen radicals are generated
(ROS), lead to oxidative stress, cause breaks down proteins, lipid peroxidation and DNA damage, promote neuronal apoptosis.And
And oxidative stress leads to the activation of microglia and astroglia, discharges a large amount of inflammatory factors, promotes blood-spinal cord screen
The destruction of barrier leads to edema of spinal cord and neutrophil infiltration after ischemia of spinal cord.Therefore, blocking oxide stress and subsequent inflammation
Disease is reacted and Apoptosis access is the main thought of spinal nerve protection treatment.
Clinically, Reperfusion Injury of Spinal Cord (Spinal cord ischemia-reperfusion injury,
SCIRI) often merging or secondary in spine and spinal cord wound lesion, rare independent generation.Common method has surgical operation, drug
Treatment, gene therapy, transplantation treatment etc., this adds increased treatment costs, increase the burden of patient.
In view of this, special propose the present invention.
Invention content
The first object of the present invention is to provide the preparation method of a kind of over-saturation methane physiological saline and obtained super
It is saturated methane physiological saline, which prepares simply, and in obtained over-saturation methane physiological saline
Methane content it is high.
The second object of the present invention is to provide a kind of over-saturation methane physiological saline and treats activation core preparing
Application in factor E2 correlation factors -2 (Nrf2) signal path drug, especially in Reperfusion Injury of Spinal Cord drug
Using.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
Sterile saline and methane gas be by a kind of preparation method of over-saturation methane physiological saline according to volume ratio
3：0.8-1.2 input internal layers are covered in the aluminium foil bag of polyethylene film, until the pressure in the aluminium foil bag reaches 0.18-
0.22MPa；
The aluminium foil bag is handled 6-8 hours under the pressure of 0.45-0.55MPa；
Then methane gas is inputted in the aluminium foil bag with complement lysis in the part of physiological saline, makes the aluminium foil bag
Interior pressure is maintained at 0.18-0.22MPa.
In recent years it has been reported that methane has organ protection.However, methane gas is in room temperature (15-25 DEG C) normal pressure
In (1 atmospheric pressure, i.e. 0.1MPa) environment, solubility in water is smaller, only 0.22mmol/L, it is difficult to reach clinical treatment
Required concentration.Therefore it is high containing methane concentration to develop one kind, it is easy to carry, it is clinically easy to use, store, and can protect for a long time
The dichloromethane for holding its titer plateaus is very necessary.
The preparation method of over-saturation methane physiological saline provided by the invention, specific selection internal layer are covered with polyethylene film
Aluminium foil bag inputs physiological saline and methane gas as carrier according to a certain percentage, until reach certain pressure in aluminium foil bag,
Then it handles under pressure, using extra injection methane gas, with complement lysis in the part of physiological saline so that
Methane gas is dissolved in the level for reaching in physiological saline and tending to over-saturation or saturation.This method uses high pressure dissolving technology, knot
Close aluminium foil bag sealed pressure storage technology, methane is dissolved into over-saturation state in physiological saline, improve methane room temperature, often
The solubility of pressure, reaches 2.50-2.65mmol/L, is 11.7 times under usual state, and can keep this concentration stablize 1 year with
On.
Wherein, sterile saline and methane gas are 3 according to volume ratio：0.8-1.2 input internal layers are covered with polyethylene
In step in the aluminium foil bag of film, the volume ratio of sterile saline and methane gas can be 3：0.8,3：1,3：1.2 etc..
For the ease of operation, and methane gas is made preferably to reach over-saturation state with physiological saline, further, institute
The volume ratio for stating sterile saline and the methane gas is 3：1.
Further, the aluminium foil bag first sterilizes before, and the sterilizing is carried out using gamma-rays irradiation.Irradiation
Sterilizing is a kind of effective ways that the electromagnetic wave generated using ionising radiation kills the microorganism on most of substances.Gamma-rays shines
It is most common irradiation sterilization technology to penetrate sterilizing, generally generates gamma-rays as radioactive source using Co 60, to play disinfection, sterilizing
Effect.
Preferably, the aluminium foil bag, which is placed in high-pressure chamber, carries out pressure treatment, simple operation.
In order to make methane gas more fully dissolve in physiological saline, it is preferable that pressure of the aluminium foil bag in 0.5MPa
Lower processing 7-8 hours.
Further, the purity of the methane gas is 99.9% or more.
The preparation method of over-saturation methane physiological saline provided by the invention, has the following advantages：
1, preparation method is simple.According to methane under normal temperature environment, solubility in water and the proportional original of pressure
Reason, by increasing ambient pressure, to increase the amount that methane dissolves in water, to reach clinical required treatment concentration.
2, over-saturation methane physiological saline potency obtained is lasting.This technology pastes the aluminium of layer of polyethylene film using internal layer
Foil bag is as liquid storage container, that is, maintaining the customary storage conditions of medical liquid medicine, (medical infusion bags generally do material with polyethylene film
Material), and overcome the micro-molecular gas such as methane and readily penetrate through the drawbacks of conventional medical infusion bags the generate potency decaying (aluminium of outer layer
Layers of foil is not easy to be passed through by micro-molecular gas).In addition, aluminium foil bag, due to being metal quality, more conventional medical polyethylene bag can
Larger tension is born, makes to maintain higher pressure (0.2MPa) in bag, to maintain over-saturation of the methane in physiological saline
The stabilization of dissolving.
3, this technology only needs to store under room temperature, and overcoming methane injection that conventional art obtains need to be under 0-5 DEG C of environment
The harsh conditions of preservation.
A concentration of 2.50-2.65mmol/L of methane in over-saturation methane physiological saline made from the above method of the present invention.
Nuclear factor Nrf2 signal paths are the presently found anti-oxidant responsing reactions for resisting external source sexual stimulus and poisonous substance
Core transcription factor.The inventors discovered that over-saturation methane physiological saline has the function of activating nuclear factor Nrf2 signal paths,
So that organism is to anti-oxidant generation response.
Further, the present invention provides over-saturation methane physiological saline to treat activation Nrf2 signal paths as preparation
Application in drug.
Specifically, the over-saturation methane physiological saline is preparing answering in treating Reperfusion Injury of Spinal Cord drug
With.
The therapy of Reperfusion Injury of Spinal Cord, including give a effective amount of over-saturation methane physiology salt of subject
Water.
Further, based on the weight of organism, the dosage of the over-saturation methane physiological saline is 15-25ml/
kg。
Preferably, the dosage of the over-saturation methane physiological saline is 20ml/kg.
Over-saturation methane physiological saline provided by the invention is for treating Reperfusion Injury of Spinal Cord.
Compared with prior art, beneficial effects of the present invention are：
(1) preparation method of over-saturation methane physiological saline provided by the invention, using high pressure dissolving technology, in conjunction with aluminium foil
Bag sealed pressure storage technology, methane is dissolved into over-saturation state in physiological saline, improves methane under room temperature, normal pressure
Solubility reaches 2.50-2.65mmol/L, is 11.7 times under usual state, and this concentration can be kept to stablize 1 year or more.
(2) experiment is found, over-saturation methane physiological saline provided by the invention has effects that activate Nrf2 signal paths.
(3) the present invention also provides over-saturation methane physiological saline is applied to treatment Reperfusion Injury of Spinal Cord, tool
There is fine effect.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is over-saturation methane physiological saline obtained in the embodiment of the present invention 1 with the extension methane concentration of holding time
Change curve；
Fig. 2 is the different other rat hindlimb movement-sensory disturbance index curve diagrams of group in the embodiment of the present invention 2；
Fig. 3 is the different other In Rat Lumbar section ventricornu normal neurons quantity column diagrams of group in the embodiment of the present invention 2；
Fig. 4 is the different methane concentration curve graphs organized in other rat plasma, myeloid tissue in the embodiment of the present invention 2；
Fig. 5 is the different schematic diagrames for organizing other rat experiment flow and index determining in the embodiment of the present invention 3；
Fig. 6 is the different result figures for organizing other rat hindlimb nervous function and tectology in the embodiment of the present invention 3.
Fig. 7 is that difference organizes other rat serum-spinal-cord barrier permeability, spinal cord water content and neutrality in the embodiment of the present invention 3
The result figure of granulocyte infiltration；
Fig. 8 is the different result figures for organizing other In Rat Lumbar section spinal cord methane concentration in the embodiment of the present invention 3；
Fig. 9 is the different other rat nuclear factor Nrf2 protein expression situation schematic diagrams of group in the embodiment of the present invention 3；
Figure 10 is that difference organizes the various antioxidases of other rat and oxidative injury markers variation in the embodiment of the present invention 3
Figure；
Figure 11 is Nrf2 albumen in the different other rats of group in the embodiment of the present invention 3 in neuron expression situation map；
Figure 12 is that difference organizes other rat No microglial state of activation and Nrf2 albumen small in the embodiment of the present invention 3
Spongiocyte expression figure；
Figure 13 is that star-like Glial Activation state and Nrf2 albumen exist in the different other rats of group in the embodiment of the present invention 3
Star spongiocyte expression figure；
Figure 14 is that spinal neuron tune dies situation map in the different other rats of group in the embodiment of the present invention 3；
Figure 15 is the different marker variations organized other neurons of rats mitochondria tune and die approach in the embodiment of the present invention 3
Figure；
Figure 16 is that difference organizes other rat proinflammatory factor, chemotactic factor (CF), adhesion molecule and MMP-9 in the embodiment of the present invention 3
Expression figure.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Prepare over-saturation methane physiological saline：
The aluminium foil bag that internal layer is covered with one layer of non-toxic polyethylene film is selected to be held by 500ml as liquid storing bag and infusion bag
Long-pending bag body (is prepared) with infusion connector composition by Suzhou City of Jiangsu Province stars new material Co., Ltd, and infusion connector is close by rubber
Seal seals；Gamma-rays radiation sterilization is used using preceding；
By sterile saline, methane gas (purity 99.9%) with 3:1 volume ratio injects aluminium foil by infusion connector
In bag, fill until pressure reaches 0.2MPa (from infusion connector, by being inserted in infusion connector rubber seal in aluminium foil bag
Enter one end and be connected with No. 6 infusion niidls of pressure gauge to measure)；
The aluminium foil bag merging hyperbaric chamber that will be filled with methane, makes pressure in hyperbaric chamber rise to 0.5MPa, and maintain this pressure up to 8
Hour, methane gas is dissolved in physiological saline and reaches over-saturation level；
Aluminium foil bag is taken out from hyperbaric chamber, then a small amount of methane gas is inputted from infusion connector, with complement lysis in physiology
The part of brine makes a bag internal pressure try hard to keep and holds 0.2MPa, is subsequently placed under room temperature and preserves.
Concentration containing methane in the over-saturation methane physiological saline prepared is measured using gas chromatography, obtains fresh system
Methane mean concentration in standby 1 day physiological saline is 2.63mmol/L；After preparing January, the methane in physiological saline is average dense
Degree is 2.61mmol/L, and after preparing 1 year, the methane mean concentration in physiological saline remains at 2.54mmol/L, specific such as Fig. 1
It is shown.
Embodiment 2
Spinal cord ischemia/reperfusion model starts to be injected intraperitoneally respectively at once in embodiment 1 obtained in Reperfu- sion
Over-saturation methane physiological saline, based on the weight of rat, injection volume is respectively 5ml/kg, 10ml/kg and 20ml/kg, respectively
Methane-5 groups, Methane-10 groups, Methane-20 groups, while blank control group is set, i.e. IR groups；
Reperfu- sion rat hindlimb movement in 72 hours-sensory disturbance index (Neurological deficits), waist section ridge are measured respectively
Marrow anterior angle normal neurons quantity and blood plasma and Yao Duan myeloid tissues methane concentration.Rat hindlimb movement-sensory disturbance index
The results are shown in Figure 2, and the results are shown in Figure 3 for waist section ventricornu normal neurons quantity, blood plasma and Yao Duan myeloid tissues methane
Concentration results are as shown in Figure 4.In Fig. 3, A represents IR groups, and B represents Methane-5 groups, and C represents Methane-10 groups, and D is represented
Methane-20 groups, arrow indicate normal neurons；E is 4 groups of normal neurons quantity statistics.
Experimental result is shown, compared with ischemia-reperfusion group (IR groups), 20ml/kgMS (Methane-20 groups) is injected intraperitoneally
The methane concentration being remarkably improved in blood plasma, myeloid tissue improves movement-sensory disturbance, preserves ventricornu normal neurons；
And injection volume is 5ml/kg then little on improving movement-sensory disturbance influence；Movement-sensory disturbance when injection volume is 10ml/kg
Slightly improve.
It is obtained from above-mentioned experiment, over-saturation methane physiological saline amount, which is 20ml/kg, can realize treatment effect well
Fruit.
In addition, also using the over-saturation methane physiological saline dosage of 15ml/kg and 25ml/kg, the injection of 15ml/kg
The injection dosage effect of dose effect ratio 20ml/kg is slightly worse, and the over-saturation methane physiological saline dose effect of 25ml/kg
Suitable with the injection dosage effect of 20ml/kg, still, due to the limitation of rat abdominal cavity size, more multi-dose causes the difficulty of injection
Degree.It is therefore preferable that using the over-saturation methane physiological saline dosage of 20ml/kg.
Embodiment 3
Rat is randomly divided into five groups, respectively sham groups, IR groups, methane groups, Si-Methane groups and Con-
Methane groups, every group 38.Specific every group of information is as follows：
Sham groups：Sham-operation group, except Reperfusion Injury of Spinal Cord is not carried out, other are the same as IR groups.
IR groups：It is prepared using the method for blocking aorta pectoralis to restore perfusion and blood pressure after merging body circulation low blood pressure 9 minutes
Spinal cord ischemia/reperfusion model.
Methane groups：Using the method for blocking aorta pectoralis to restore perfusion and blood pressure after merging body circulation low blood pressure 9 minutes
Prepare spinal cord ischemia/reperfusion model；Over-saturation methane physiological saline (MS) is injected intraperitoneally at once in restoring perfusion
20ml/kg。
Si-Methane groups：300 μ g/kg of rat intrathecal injection Nrf2 siRNAs (Nrf2 siRNA), every 24 hours
Injection is primary, continuous 3 times.After last time intrathecal injection 24 hours, implement operation, merges body circulation using aorta pectoralis is blocked
Low blood pressure restores perfusion after 9 minutes and the method for blood pressure prepares spinal cord ischemia/reperfusion model, is in restoring perfusion
Carve intraperitoneal injection over-saturation methane physiological saline (MS) 20ml/kg.
Con-Methane groups：300 μ g/kg of rat intrathecal injection negative control siRNA (control siRNA), often
It is primary every injection in 24 hours, continuous 3 times.After last time intrathecal injection 24 hours, implement operation, is closed using aorta pectoralis is blocked
And body circulation low blood pressure restores perfusion after 9 minutes and the method for blood pressure prepares spinal cord ischemia/reperfusion model, Yu Hui
Over-saturation methane physiological saline (MS) 20ml/kg is injected intraperitoneally in multiple perfusion at once.
The difference of specific each group is as shown in Figure 5.
1, MS can improve IR injury rats hind leg nervous functions and tectology
Hind leg Neurological deficits (movement-sensory disturbance is carried out to each group rat in Reperfu- sion 12,24,48,72 hours
Index, Fig. 6 A)；Nissl dyeing, observation morphological change (figure were carried out to each group Rat Lumbar Ventral Spinal Cord in 72 hours in Reperfu- sion
6B), the normal neurons quantity (Fig. 6 C) of ventricornu, middle part and relief angle is measured.
Each group rat can survive to Reperfu- sion after 72 hours, to ensure the neurogenic behavior rating of Each point in time.Such as
Shown in Fig. 6 A, 9 minutes ischemias of spinal cord can cause the apparent neuroethology obstacle of rat；Compared with Sham groups, IR groups in filling again
12,24,48,72 hourly average campaign of note-sensory disturbance index obviously increases (p<0.01)；Compared with IR groups, Methane groups are each
Time point mean motion-sensory disturbance index is decreased obviously (p<0.01)；Compared with Methane groups and Con-Methane groups,
Each time point mean motion-sensory disturbance index of Si-Methane groups obviously increases (p<0.05)；Compared with Methane groups,
Each time point mean motion-sensory disturbance index differential of Con-Methane groups is not statistically significant (p > 0.05)
9 minutes ischemias of spinal cord can cause rat spinal cord grey matter each region neuronal death.As shown in Fig. 6 B, C：With Sham
Group compares, and IR groups ventricornu, middle part, relief angle normal neurons quantity are remarkably decreased (p<0.001)；Compared with IR groups,
Each region normal neurons quantity of Methane group spinal cords obviously increases (p<0.01)；With Methane groups and Con-Methane groups
Compare, each region normal neurons quantity of Si-Methane group spinal cords is decreased obviously (p<0.05)；Compared with Methane groups,
Each region normal neurons quantity variance of Con-Methane group spinal cords is not statistically significant (p > 0.05).
2, MS mitigates IR injury rats blood-spinal cord barriers permeability, spinal cord water content and neutrophil infiltration
Each group In Rat Lumbar section spinal cord Evans blue fluorescence intensity (Fig. 7 A, B) and content (figure are measured within 72 hours in Reperfu- sion
7C) (the two all reflects blood-spinal cord barrier permeability), spinal cord water content (Fig. 7 D) and activity of myeloperoxidase (Fig. 7 E, reflection
Neutrophil infiltration degree).
As can be seen from Figure 7, compared with Sham groups, IR groups waist section spinal cord Evans blue content and fluorescence intensity, water content, marrow mistake
Peroxidase activity dramatically increases (p<0.01)；Compared with IR groups, Methane groups spinal cord Evans blue content and fluorescence intensity,
Water content, activity of myeloperoxidase significantly reduce (p<0.01 or p<0.05)；With Methane groups and Con-Methane group ratios
Compared with Si-Methane groups spinal cord Evans blue content and fluorescence intensity, water content, activity of myeloperoxidase significantly increase (p<
0.05)；Compared with Methane groups, Con-Methane groups spinal cord Evans blue content and fluorescence intensity, water content, marrow peroxidating
Object enzymatic activity no significant difference (p > 0.05).
3, gas chromatography measures each group In Rat Lumbar section spinal cord methane concentration
Fig. 8 A are chromatography of gases figures, reflection Methane groups be injected intraperitoneally 10 minutes (a1) after 20ml/kg MS, 12 hours
(a2), 24 hours (a3), 48 hours (a4), 72 hours (a5) myeloid tissue methane concentrations.Fig. 8 B are each time point waist of each group rat
The comparison of section spinal cord methane concentration.
From figure 8, it is seen that compared with Sham groups and IR groups, Methane groups, Si-Methane groups, Con-Methane groups
10min spinal cords methane concentration significantly increases (p< after intraperitoneal injection MS;0.001), and 12 after administration, holding in 24,48,72 hours
Higher methane concentration (p<0.01).In each time point ridge between Methane groups, Si-Methane groups, three groups of Con-Methane groups
Marrow methane concentration no significant difference (p > 0.05).IR groups, though ischemic can cause methane concentration in spinal cord slightly to increase,
But its concentration and Sham group comparing differences are not statistically significant (p > 0.05).
4, MS treats the increase of Nrf2 protein expressions and its nuclear translocation after induced myeloid IR damages
Inventor has found that MS treatments mitigate spinal cord IR damage, increase with MS induction nuclear factor Nrf2 protein expressions and
Promote its nuclear translocation related, and is in time dependence.
Fig. 9 A-D are shown, compared with Sham groups, IR groups in Reperfu- sion 12,24,48,72 hours Nrf2 albumen nucleus,
Cytoplasmic expression quantity and nucleus/cytoplasmic proportional difference are not statistically significant (p > 0.05)；Compared with IR groups,
Each time point spinal cord Nrf2 albumen of Methane groups is in nucleus (p<0.05vs.IR for 12,24h,p<0.01vs.IR for
48,72h), cytoplasm (p<0.05vs.IR for 12,24,48h,p<0.01vs.IR for 72h) in expression quantity and cell
Ratio (the p< of matter/nucleus;0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h) it gradually increases；With
Methane groups and Con-Methane groups compare, and each time point Nrf2 albumen of Si-Methane groups is in nucleus, cytoplasm
Expression quantity and nucleus/cytoplasmic ratio are substantially reduced (p<0.05)；Compared with Methane groups, Con-Methane groups Nrf2
Albumen is not statistically significant (p > 0.05) in nucleus, cytoplasmic expression quantity and nucleus/cytoplasmic proportional difference.
Fig. 9 E-F are shown, compared with Sham groups, IR groups are in Reperfu- sion 12,24,48,72 hours each time point Nrf2 mRNA
Expression quantity, DNA binding activity no significant difference (p > 0.05)；Compared with IR groups, each time point spinal cord of Methane groups
Nrf2 mrna expression amounts (p<0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h) and its DNA binding activity
(p<0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h) it gradually increases；With Methane groups and Con-
Methane groups compare, and each time point Nrf2 mrna expression amounts of Si-Methane groups, DNA binding activity decline (p<0.05)；With
Methane groups compare, Con-Methane group Nrf2 mrna expression amounts, DNA binding activity no significant difference (p >
0.05)。
5, the anti-oxidation protection mechanism that Nrf2 regulates and controls after the IR damages of MS treatments activation spinal cord
On the basis of the above result of study, whether the Antioxidation Mechanism that inventor further inquires into Nrf2 regulation and control takes part in
The cord dorsal potentials of MS act on, and mitigate oxidative damage.
Figure 10 A-D are shown, compared with Sham groups, IR groups are in Reperfu- sion 12,24,48,72 hours Protoheme oxygendase-1 eggs
White expression difference is not statistically significant (p > 0.05), and superoxide dismutase, catalase activity are gradually reduced (p<
0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h).Compared with IR groups, Methane groups in Reperfu- sion 12,
24,48,72 hours Protoheme oxygendase-1 albumen and superoxide dismutase, catalase activity gradually increase (p<
0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h).Compared with Methane groups and Con-Methane groups,
Each time point HO-1 albumen of Si-Methane groups and superoxide dismutase, catalase activity are substantially reduced (p<0.05).
Compared with Methane groups, each time point Protoheme oxygendase-1 albumen of Con-Methane groups and superoxide dismutase, peroxide
It is not statistically significant (p > 0.05) to change hydrogenase activity activity difference.
Figure 10 E-G are shown, compared with Sham groups, IR groups are de- in Reperfu- sion 12,24,48,72 hours spinal cord malonaldehyde, 8- hydroxyls
Oxygen guanosine, 3- nitrotyrosines content significantly increase (p<0.05vs.IR for 12h,p<0.01vs.IR for 24,48,
72h).Compared with IR groups, each time point malonaldehyde of Methane groups, 8- hydroxyls deoxyguanosine, 3- nitrotyrosine contents continuously decrease
(p<0.05vs.IR for 12,24h,p<0.01vs.IR for 48,72h).With Methane groups and Con-Methane group ratios
Compared with each time point malonaldehyde of Si-Methane groups, 8- hydroxyls deoxyguanosine, 3- nitrotyrosine contents dramatically increase (p<0.05).
Compared with Methane groups, Con-Methane groups malonaldehyde, 8- hydroxyls deoxyguanosine, 3- nitrotyrosine content differences are without statistics
Meaning (p > 0.05).
6, in neuron, microglia, star spongiocyte expression after the IR damages of MS treatments induction Nrf2 albumen spinal cords
Figure 11 A, 12C, 13C show Reperfu- sion 72 hours Nrf2 albumen respectively with neuron (NeuN), microglia
(Iba-1), star spongiocyte (GFAP) Double immunofluorescence dyes.
Experimental result：Sham group In Rat Lumbar section gray nucleus is only shown in the fluorescence intensity of a small amount of Nrf2 protein expressions, and its
Most of cytoplasms for being distributed in neuron, microglia, star spongiocyte.The fluorescence of IR group gray nucleus Nrf2 albumen is strong
Degree indifference (p > 0.05) compared with Sham groups with cell distribution.Methane group gray nucleus anterior angle, middle part, relief angle all may be used
See a large amount of Nrf2 protein immunizations fluorescence (p < 0.01vs.IR), and is largely distributed in neuron, microglia, star-like glue
The karyon of cell plastid.Si-Methane group gray nucleus anterior angle, middle part, relief angle Nrf2 protein immunizations fluorescence intensity compared with
Methane groups, Con-Methane groups substantially reduce (p<, and most of neuron, microglia, star-like of being distributed in 0.05)
The endochylema of spongiocyte.The Nrf2 fluorescence intensities in Con-Methane group gray nucleus region, distribution (p similar to Methane groups
> 0.05).Figure 11 B, 12D, 13D are to be co-expressed with neuron, microglia, star spongiocyte in 5 groups of gray nucleus
The comparison of Nrf2 protein fluorescence intensity.
7, neuron tune is died after MS treatments mitigate spinal cord IR damages
Figure 14 shows that the dUTP of Reperfu- sion each group waist section spinal cord progress terminal deoxynucleotidyl transferase mediation in 72 hours is lacked
Mouth end mark (TUNEL), reflection spinal neuron tune die situation.
Sham groups spinal cord is only shown in few TUNEL positive cells；The visible a large amount of TUNEL of IR groups ventricornu, middle part, relief angle
Positive cell (p < 0.001vs.Sham).Compared with IR groups, Methane groups ventricornu, middle part, relief angle TUNEL are positive thin
Born of the same parents substantially reduce (p < 0.01).Si-Methane group TUNEL positive cells obviously increase compared with Methane groups, Con-Methane groups
Add (p < 0.05).Compared with Methane groups, Con-Methane group TUNEL cell number no statistical difference differences (p >
0.05)。
Figure 15 shows that 72 hours MS of Reperfu- sion die neuron linear plastochondria tune the marker cytochrome c and activated form of approach
The influence of Caspase-9, activated form caspase-3 mRNA.
Experimental result is shown：IR groups cord cell pigment c and activated form Caspase-9, activated form caspase-3 mRNA
Expressing quantity and activated form caspase-3 mRNA positive neuron quantity are significantly raised (p < 0.001vs.Sham).With IR groups
Compare, Methane groups cord cell pigment c and activated form Caspase-9, activated form caspase-3 mRNA expressing quantity
And activated form caspase-3 mRNA positive neuron quantity significantly reduces (p < 0.01).With Methane groups and Con-Methane
Group compares, Si-Methane groups cytochrome c and activated form Caspase-9, activated form caspase-3 mRNA expressing quantity
And activated form caspase-3 mRNA positive neuron quantity obviously increases (p < 0.05).Compared with Methane groups, Con-
Methane groups cytochrome c and activated form Caspase-9, activated form caspase-3 mRNA expressing quantity and activated form half
- 3 positive neuron quantity variance of Guang aspartase is not statistically significant (p > 0.05).
8, MS treatments inhibit microglia, star spongiocyte activation after spinal cord IR damages
Figure 12 A show, 72 hours waist section spinal cord slice microglia marker Iba-1 immunofluorescence dyeings of Reperfu- sion,
MS is prompted to inhibit Activated Microglia after spinal cord IR damages.
Experimental result：The visible gray nucleus of Sham groups is sporadicly dispersed with a small amount of microglia (Iba-1 positive cells), is in
Quiescent condition：Typical cellule body, there is several tiny branches.IR group gray nucleus anterior angle, middle part, relief angle microglia
Quantity showed increased (p < 0.001vs.Sham) is in the state of activation：Cell body becomes loose, and branch shortens, is thicker.With IR group ratios
Compared with Methane group gray nucleus anterior angle, middle part, relief angle microglia significantly reduce (p < 0.01), and the state of activation reduces.
Compared with Methane groups, Con-Methane groups, Si-Methane group gray nucleus anterior angle, middle part, relief angle microglia
Quantity increases (p < 0.05).Compared with Methane groups, Con-Methane group microglias quantity, the similar (p of the state of activation
> 0.05).
Figure 13 A show that 72 hours waist section spinal cord slice astrocytes marker GFAP immunofluorescence dyeings of Reperfu- sion prompt
Star spongiocyte activates after MS inhibits spinal cord IR damages.
Experimental result：The a small amount of star spongiocyte of the visible gray nucleus of Sham groups (GFAP positive cells).IR groups spinal cord ash
Matter anterior angle, middle part, relief angle star spongiocyte proliferation, quantity showed increased (p < 0.001vs.Sham).Compared with IR groups,
Methane group gray nucleus anterior angle, middle part, relief angle star spongiocyte significantly reduce (p < 0.01).With Methane groups,
Con-Methane groups compare, and Si-Methane group gray nucleus anterior angle, middle part, relief angle star spongiocyte quantity increase (p
< 0.05).Compared with Methane groups, not statistically significant (the p > of Con-Methane group star spongiocyte quantity variances
0.05)。
9, MS treatments pass through proinflammatory factor, chemotactic after interfering nuclear factor Kappa B (NF- κ B) signal to inhibit spinal cord IR damages
The factor, adhesion molecule and MMP-9 expression
Figure 16 A-C show that Reperfu- sion MS treatments in 72 hours inhibit phosphorylation (the p-NF- κ B of NF- κ B p65 subunits
) and nuclear translocation p65.
Experimental result：Compared with Sham groups, IR group spinal cord p-NF- κ B p65 nucleus/cytoplasmic ratio, DNA are combined
It is active significantly to increase (p < 0.001).Compared with IR groups, Methane group p-NF- κ B p65 nucleus/cytoplasmic ratio, DNA
It is substantially reduced (p < 0.01) in conjunction with activity.Compared with Methane groups, Si-Methane group p-NF- κ B p65 nucleus/cell
The ratio of matter, DNA binding activity are significantly raised (p < 0.05), and MS is prompted to inhibit the phosphorylation and consideration convey of NF- κ B p65 subunits
Position relies on what Nrf2 increased activities were realized.Compared with Methane groups, Con-Methane group p-NF- κ B p65 nucleus/cell
The ratio of matter, DNA binding activity no significant difference (p > 0.05).
Figure 16 D-K show, tumor necrosis factor-alpha (TNF-α), the il-1 that 72 hours MS of Reperfu- sion regulate and control NF- κ B
The inhibition of β (IL-1 β), Gro-beta-T ligand 1 (CXCL1), intercellular adhesion molecule (ICAM-1).Experimental result：With Sham
Group compares, and IR group spinal cords TNF-α, IL-1 β, the mRNA of CXCL1, ICAM-1 and content significantly increase (p < 0.001).With IR groups
Compare, Methane groups TNF-α, IL-1 β, the mRNA of CXCL1, ICAM-1 and content are substantially reduced (p < 0.01).With Methane
Group compares, and Si-Methane groups TNF-α, IL-1 β, the mRNA of CXCL1, ICAM-1 and content are significantly raised (p < 0.05), Con-
Methane groups TNF-α, IL-1 β, the mRNA of CXCL1, ICAM-1 and content difference are not statistically significant (p > 0.05).
Figure 16 L-N show, inhibition of the 72 hours MS of Reperfu- sion to the NF- κ B Matrix Metalloproteinase-9s (MMP-9) regulated and controled,
And the influence to tight junction protein (Claudin-5, Occludin, ZO-1).Experimental result：Compared with Sham groups, IR groups
Spinal cord MMP-9 expressing quantities significantly increase, and the expressing quantity of Claudin-5, Occludin, ZO-1 are substantially reduced (p <
0.001).Compared with IR groups, Methane group spinal cord MMP-9 expressing quantities are decreased obviously, Claudin-5, Occludin, ZO-
1 expressing quantity obviously increases (p < 0.01).Compared with Methane groups, Si-Methane group MMP-9 protein contents obviously increase
Adding, the protein content of Claudin-5, Occludin, ZO-1 are decreased obviously (p < 0.05), Con-Methane groups MMP-9,
The expressing quantity no significant difference (p > 0.05) of Claudin-5, occludin, ZO-1.
In addition, the preparation method that the present invention also changes the over-saturation methane physiological saline of embodiment 1 prepares over-saturation methane
Physiological saline：
Group 1：The volume of sterile saline and the volume ratio of methane gas are 3:0.8, until the pressure in aluminium foil bag reaches
0.18MPa is handled 6 hours under the pressure of 0.45MPa, and the methane concentration in obtained over-saturation methane physiological saline is
2.55mmol/L；Room temperature preserves 1 year, and methane concentration is 2.50mmol/L or more.
Group 2：The volume of sterile saline and the volume ratio of methane gas are 3:1.2, until the pressure in aluminium foil bag reaches
0.22MPa is handled 8 hours under the pressure of 0.55MPa, and the methane concentration in obtained over-saturation methane physiological saline is
2.65mmol/L；Room temperature preserves 1 year, and methane concentration is 2.60mmol/L or more.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (9)
1. a kind of preparation method of over-saturation methane physiological saline, which is characterized in that press sterile saline and methane gas
It is 3 according to volume ratio：0.8-1.2 input internal layers are covered in the aluminium foil bag of polyethylene film, until the pressure in the aluminium foil bag reaches
0.18-0.22MPa；
The aluminium foil bag is handled 6-8 hours under the pressure of 0.45-0.55MPa；
Then methane gas is inputted in the aluminium foil bag with complement lysis in the part of physiological saline, is made in the aluminium foil bag
Pressure is maintained at 0.18-0.22MPa.
2. the preparation method of over-saturation methane physiological saline according to claim 1, which is characterized in that the sterile physiological
The volume ratio of brine and the methane gas is 3：1.
3. the preparation method of over-saturation methane physiological saline according to claim 1, which is characterized in that the aluminium foil bag exists
It is first sterilized using preceding, the sterilizing is carried out using gamma-rays irradiation.
4. the preparation method of over-saturation methane physiological saline according to claim 1, which is characterized in that the aluminium foil bag is put
It sets and carries out pressure treatment in high-pressure chamber.
5. the preparation method of over-saturation methane physiological saline according to claim 1, which is characterized in that the methane gas
Purity be 99.9% or more.
6. over-saturation methane physiology made from the preparation method of claim 1-5 any one of them over-saturation methane physiological saline
Brine, which is characterized in that a concentration of 2.50-2.65mmol/L of methane in the over-saturation methane physiological saline.
7. the over-saturation methane physiological saline described in claim 6 is preparing answering in treating Reperfusion Injury of Spinal Cord drug
With.
8. application according to claim 7, which is characterized in that the dosage of the over-saturation methane physiological saline is
15-25ml/kg。
9. application according to claim 8, which is characterized in that the dosage of the over-saturation methane physiological saline is
20ml/kg。
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| CN103690481A (en) * | 2013-12-23 | 2014-04-02 | 中国人民解放军第二军医大学 | Application of methane injection in preparation of medicaments for treating ischemia-reperfusion injury |
| CN105079025A (en) * | 2015-06-17 | 2015-11-25 | 泰山医学院 | Application of hydrogen saturated saline |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103690481A (en) * | 2013-12-23 | 2014-04-02 | 中国人民解放军第二军医大学 | Application of methane injection in preparation of medicaments for treating ischemia-reperfusion injury |
| CN105079025A (en) * | 2015-06-17 | 2015-11-25 | 泰山医学院 | Application of hydrogen saturated saline |
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