CN105250246B - Application of the protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm - Google Patents
Application of the protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm Download PDFInfo
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Abstract
The present invention relates to a kind of protocatechualdehyde to prepare the application in preventing and treating caused by cyclophosphamide male sterility disease drug, belongs to treatment male sterility technical field.Technical scheme is：Protocatechualdehyde is used for male sterility and Reproductive Damage caused by preventing and treating endoxan.Present invention treatment male sterility, relative low price, can be promoted the use of.Protocatechualdehyde, which removes, has anti-oxidative stress, moreover it is possible to improves the protein contents of testis DJ 1, is better than carnitine and vitamin E in treatment male sterility mechanism.
Description
Technical field
The present invention relates to a kind of application of protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm, particularly original
Application of the catechu aldehyde in the male sterility as caused by endoxan is treated, and protocatechualdehyde are led for preventing and treating endoxan
The application of the Male reproductive damage of cause, belong to treatment male sterility and extracorporeal treatment sperm technical field.
Background technology
With industrialized development, the aggravation of environmental pollution and the increase of social pressures, male semen quality year by year under
Drop, male sterility patient substantially increase.By the World Health Organization（World Health Organization, WHO）Standard,
The couple at child-bearing age lives together more than 1 year after marriage, not using any contraceptives, causes the infertile person in the wife's side, referred to as male by bridegroom's or husband's side reason
Sterility.The treatment of male sterility mainly includes Issues of Human Assisted Reproductive Technologies, drug therapy improves sperm quality, and operation
Treatment correction physiological structure is abnormal.Cause the cause of disease of male sterility sufficiently complex, not yet find causes male sterility at present
The definite cause of disease, so being mainly empirical drug therapy to male sterility.The conventional medical treatment regime of clinic has：Antioxygen
Change treatment, including conventional vitamin E, Co-Q10 etc.；Hormone therapy, including antiestrogenic, promoting sexual gland hormone, rush sexual gland swash
Hormone-releasing hormone, androgen, Aromatase inhibitor；Other drugs, including carnitine etc..But these medicines have respective office
It is sex-limited, it is such as expensive, side reaction is more, therefore researching and developing new male sterility medicine has important clinical meaning
Be widely applied prospect.
Issues of Human Assisted Reproductive Technologies can be used for treatment male sterility and female infertility, including artificial insemination and external
Fertilization-embryo transfer technology, two kinds of technologies are required for carrying out the preferred sperm of extracorporeal treatment to semen.Sperm culture is excellent
It is the conventional required step in Issues of Human Assisted Reproductive Technologies treatment to select sperm, it is therefore an objective to obtains no refining, cell fragment and disease
The sperm suspension of pathogenic microorganism pollution, recovery sufficient amount have the sperm of normal morphology and function.Needed during liquefacient duration
Sperm nutrient solution is added to maintain sperm motility and function.At present, the sperm nutrient solution used in background technology still has much room for improvement, I
Used in state is mostly imported product, expensive.Exploitation can improve the sperm nutrient solution composition of sperm motility for sperm body
Preferably there is key effect and significance with raising supplementary reproduction Clinical Pregnancy Rate in outside.
Protocatechualdehyde（Protocatechuic aldehyde, PAL）Scientific name 3,4- 4-dihydroxy benzaldehydes, it is that the red sage root is water-soluble
Property the degraded of composition tanshin polyphenolic acid B one of primary product, be present in the plants such as the labiate red sage root, exist in Lindsaeaceae
In the leaf of plant Stenoloma chusana, in holly plant Folium Ilicis Purpureae, source is quite varied.Research shows that protocatechualdehyde has very wide
General pharmacological activity, including it is antiatherosclerosis, protection cardiac muscle, antithrombus formation, neuroprotection, anti-purulence blood, antiviral, anti-
Fibrosis etc..So far, the purposes about protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm does not have also
There is document report.
The content of the invention
It is an object of the present invention to provide a kind of application of protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm, solution
Certainly above mentioned problem present in background technology.
The technical scheme is that：
Application of the protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm, protocatechualdehyde is used to treat male
Sterility and extracorporeal treatment sperm.
The protocatechualdehyde is used to treat male sterility, and protocatechualdehyde aqueous stability is poor, using solid pharmaceutical preparation,
Such as tablet, granule, capsule, soft capsule, dripping pill.
Dosage：Converted according to zoopery, recommended dose is 4.4 mg/kg（About 300 mg/d）.
Administrated method：Orally.
Drug treatment：Recommend 1-2 spermatogenic cycle, i.e. individual month of 3-6.
It can be used alone, can also be shared with other drugs.
The protocatechualdehyde is used for extracorporeal treatment sperm, and protocatechualdehyde aqueous stability is poor, and powder-injection is made, current
Now match somebody with somebody, after a small amount of physiological saline solution, add in sperm nutrient solution and use, recommended density scope is 0.1 mg/ml-1mg/
ml。
The present invention is by testing proof, the testis index of chmice in male sterile caused by protocatechualdehyde can increase endoxan
And epididymis index, Maturation of sperm nucleus is improved, protection testis tissue form is normal, increases SOD activity in testis, improves in testis
DJ-1 protein contents.Protocatechualdehyde has the pharmacological activity for the treatment of male sterility.
The present invention is proved by experiment in vitro, after protocatechualdehyde is incubated in vitro with sperm, can significantly improve sperm survival
Rate and sperm membrane integrity, there is certain application prospect in terms of handling sperm in vitro.
The treatment of male sterility mainly includes drug therapy and Issues of Human Assisted Reproductive Technologies.The curative of male sterility
Species are limited, need badly and develop new medicine.In Issues of Human Assisted Reproductive Technologies extracorporeal treatment, its institute must be carried out to sperm
The sperm nutrient solution needed is still to be improved.It is a discovery of the invention that protocatechualdehyde can be used for treatment and the sperm in vitro of male sterility
Processing, and there is unexpected technique effect.
The derivant endoxan of male sterility is clinically conventional antineoplastic and immunosupress in the present invention
Agent, effect is good and cheap, but endoxan has obvious male genetic toxicity, largely limits its application.
Present invention demonstrates that protocatechualdehyde can be used for Male reproductive damage caused by preventing and treating endoxan, so as to promote endoxan
Use clinically, or patient reduce the financial burden.
Protocatechualdehyde treats the advantages of male sterility and beneficial effect：
For male sterility, the medical treatment regime that clinic is commonly used at present has：Hormone therapy, including antiestrogenic, rush
Gonadal hormone, gonadotropin-releasing hormone (GRH), androgen, Aromatase inhibitor；Antioxidation Treatment, including conventional vitamin
E, Co-Q10 etc.；Other drugs, including carnitine etc..
（One）Hormone therapy：Antiestrogenic curative effect is not affirmed, is not suitable for long-term treatment；The effect of promoting sexual gland hormone, has
Limit, application are less；Gonadotropin-releasing hormone (GRH) is costly, and curative effect is not good enough, does not recommend typically；Androgen in treating is to pregnant
Rate of being pregnent simultaneously is not improved；Aromatase inhibitor can not improve semen quality, and expensive, therefore clinical less use.
As can be seen here, hormone therapy clinically and is of little use, and expensive；And protocatechualdehyde abundance, therefore price is relatively low
It is honest and clean, it can promote the use of.
（Two）Antioxidation Treatment：Vitamin E curative effect is clearer and more definite, and its mechanism of action is mainly anti-oxidation stress, and vitamin E is not
Water is dissolved in, edible oil is contained in preparation, treatment male sterility long-term use Vitamin E preparation may cause patient's fat intake
Increase, so as to trigger relevant disease, such as dyslipidemia；And protocatechualdehyde good water solubility, patient's fat intake will not be increased.
Co-Q10（400 mg/d, 6 months）Patient's semen quality can be improved, by drug price, about 12 yuan of patient's daily cost, improved
Semen quality（6 months）About 2160 yuan of expense；And protocatechualdehyde price is less expensive.The mechanism of action of vitamin E and Co-Q10 is
It is anti-oxidant, and protocatechualdehyde also has other mechanism of action in addition to can be to anti-oxidation stress（Increase testis DJ-1 protein contents）.
（Three）Other：For carnitine at present using wide, carnitine treatment male sterility common dose be 2-3g/d, is joined
Examine drug price, patient's daily cost 22-33 members, 1 course for the treatment of（3 months）Medical expense 1980-2970 members, 2 courses for the treatment of（6
Month）Medical expense 3960-5940 members, expense are of a relatively high；And protocatechualdehyde abundance, and it is few per consumption per day（300 mg/
d）, therefore medical expense is lower than carnitine.The mechanism of action of carnitine is mainly anti-oxidant, and protocatechualdehyde is except can be to anti-oxidant
Stress be outer, there are other mechanism of action（Increase testis DJ-1 protein contents）.In summary, clinical treatment male sterility at present
It is carnitine and vitamin E using more medicine, the main mechanism of the two is anti-oxidation stress, and protocatechualdehyde is except tool
Have outside anti-oxidative stress, moreover it is possible to improve testis DJ-1 protein contents, be better than carnitine in treatment male sterility mechanism
And vitamin E.The treatment male sterility it is therefore believed that protocatechualdehyde clinically can be used alone, also can be with carnitine
It is used in combination with vitamin E.
The advantages of protocatechualdehyde extracorporeal treatment sperm and beneficial effect：
Currently used sperm nutrient solution has Ham ' s-F10, Earle ' s, HTF etc., and its main component is various inorganic
Salt and amino acid, do not add other compositions.Protocatechualdehyde is added in sperm nutrient solution, its anti-oxidation stress work can be played
With caused oxygen radical during removing dealing sperm, so as to protect sperm morphology and function, than sperm is used alone
Nutrient solution effect is more.In addition, protocatechualdehyde is cheap, the running cost of extracorporeal treatment sperm will not be increased, and use
It is convenient（It is directly added into nutrient solution）, will not substantially increase operating procedure and time.
Brief description of the drawings
Fig. 1 is the influence for the Testis Morphology that protocatechualdehyde of the present invention causes chmice in male sterile to endoxan（×400）；
Fig. 2 is the influence for the testis tissue SOD activity that protocatechualdehyde of the present invention causes chmice in male sterile to endoxan（
± S, n=10）；
Fig. 3 is the influence for the testis DJ-1 protein expressions that protocatechualdehyde of the present invention causes chmice in male sterile to endoxan（
± S, n=3）.
Embodiment
Below in conjunction with accompanying drawing, by example, the invention will be further described.
Application of the protocatechualdehyde in treatment male sterility and extracorporeal treatment sperm, protocatechualdehyde is used to treat male
Sterility and extracorporeal treatment sperm.
The protocatechualdehyde is used to treat male sterility, uses solid pharmaceutical preparation；
Dosage：4.4 mg/kg（About 300 mg/d）；
Administrated method：Orally.
Drug treatment：Individual month of 1-2 spermatogenic cycle, i.e. 3-6.
It can be used alone, can also be shared with other medicines.
The protocatechualdehyde is used for extracorporeal treatment sperm, and powder-injection, matching while using, with a small amount of physiological saline solution is made
Afterwards, add in sperm nutrient solution and use, concentration is 0.1 mg/ml-1mg/ml.
The present invention is by testing proof, the testis index of chmice in male sterile caused by protocatechualdehyde can increase endoxan
And epididymis index, Maturation of sperm nucleus is improved, protection testis tissue form is normal, increases SOD activity in testis, improves in testis
DJ-1 protein contents.Protocatechualdehyde has the pharmacological activity for the treatment of male sterility.
The present invention is proved by experiment in vitro, after protocatechualdehyde is incubated in vitro with sperm, can significantly improve sperm survival
Rate and sperm membrane integrity, there is certain application prospect in terms of handling sperm in vitro.
The treatment of male sterility mainly includes drug therapy and Issues of Human Assisted Reproductive Technologies.The curative of male sterility
Species are limited, need badly and develop new medicine.In Issues of Human Assisted Reproductive Technologies extracorporeal treatment, its institute must be carried out to sperm
The sperm nutrient solution needed is still to be improved.It is a discovery of the invention that protocatechualdehyde can be used for treatment and the sperm in vitro of male sterility
Processing, and there is unexpected technique effect.
Specific experiment is as follows：
1 experiment material
1.1 key agents and reagent
Protocatechualdehyde, Beijing lark prestige Science and Technology Ltd., batch number：LC70N14；Syklofosfamid ampoule, Jiangsu
Permanent auspicious medical limited company, batch number：13052125；Aniline blue, Beijing Lei Gen Bioisystech Co., Ltd, product batch
Number：0409A15；Yihong, Beijing Lei Gen Bioisystech Co., Ltd, batch number：0409A15；DJ-1 antibody, U.S. santa
cruze；β-actin antibody, Shenyang all creation Bioisystech Co., Ltd；The goat antirabbit secondary antibody of HRP marks, Beijing Zhong Shan Golden Bridge
Bioisystech Co., Ltd；The super quick luminescent solutions of ECL, PAGE gel reagent preparation box, BCA protein quantification kits, Beijing
Suo Laibao Science and Technology Ltd.s；Bioengineering Research Institute is built up in SOD kits, Nanjing.
1.2 instrument
XL90 refrigerated centrifuges（German Beckman products）, MK3 ELIASAs（U.S.'s Thermo products）, IX83 microscopes
（Japanese OLYMPUS products）；Electrophoresis apparatus, transferring film instrument, gel imaging system（U.S.'s bio-rad products）.
1.3 animal
Male mice in kunming, 6-7 week old, provided by North China Polytechnics animal experimental center, credit number：SCXK
（Ji）2010-0038.In constant temperature（21~23℃）, constant humidity（45%~65%）, raise under each 12 h light and shade periodic conditions, ad lib
And drinking-water.
Experimental method
2.1 integral experiment
2.1.1 the packet and administration of mouse
40 mouse are randomly divided into 4 groups, are respectively：Control group, model group and protocatechualdehyde is low, high dose group.Model group
And protocatechualdehyde is low, the mg/kg/d of high dose group intraperitoneal injection of cyclophosphamide solution 60, continuous 5 days, control group intraperitoneal injection was given birth to
Manage salt solution；The protocatechualdehyde solution that protocatechualdehyde is low, high dose group gavage gives 10,40 mg/kg/d simultaneously, control group and mould
Type group gavage gives normal saline, totally 35 days.
2.1.2 mouse testis and epididymis assessment of indices
After the h of last dose 24, mouse is weighed and put to death, wins mouse bilateral testes and epididymis rapidly, carefully reject week
Adipose tissue and fascia tissue are enclosed, is weighed, calculates mouse testis and epididymis index.Testis index=testis weight/mouse weight, it is attached
Testis index=epididymis weight/mouse weight.
2.1.3 mouse sperm Nuclear maturity degree is evaluated
0.05 g aniline blues are weighed, add 20 ml absolute ethyl alcohols and the dissolving of 80 ml pure water, filter paper filtering is standby.Will be small
Mouse bilateral epididymal is placed in the physiological saline of 37 DEG C of pre-temperatures, washes away hair and blood, is put into the small burning for filling 1 ml physiological saline
In cup, epididymis is shredded with eye scissors, static 3-5 min, gently shaken.Filtered, suction strainer liquid smear, done with four layers of lens wiping paper
Dry, absolute ethyl alcohol fixes 10 min, dries, and aniline blue dyes 7 min, is capped slide, and light microscopic 400 × observation after drying is ripe
Sperm head coloring is shallow, and non-mature sperm head is navy blue.Count the percentage of mature sperm in 200 sperms.
2.1.4 mouse testis morphological observation
Mouse left testes are taken to be fixed in 10% neutral formalin, FFPE, serial section, conventional H E dyeing.Under microscope
Observe and take pictures.
2.1.5 mouse testis SOD assays
Mouse testis tissue shreds, and is homogenized in physiological saline, and 3,000 4 DEG C of rpm centrifuge 10 min, take supernatant, strictly
SOD activity and protein content in mouse testis tissue are determined according to kit specification.
2.1.6 mouse testis DJ-1 Protein Detections
Mouse testis tissue shreds, and is homogenized in RIPA lysates, and 12,000 4 DEG C of rpm centrifuge 20 min, take supernatant, sternly
Lattice determine mouse testis tissue total protein content according to kit specification, determine albumen applied sample amount.
Gel is configured according to kit specification, protein sample adds 5 × sample-loading buffer, and 100 DEG C are boiled 5 min, are made
Protein denaturation.By protein quantification result loading, the h of 50 V constant pressures electrophoresis 0.5, change voltage after albumen enters concentration glue,
The h of 100 V constant pressures electrophoresis 2.
Gel is placed in transferring film liquid and balances 30 min by electrophoresis after terminating, and pvdf membrane activates 1 min in methyl alcohol, according to
Glue and pvdf membrane are placed in membrane-transferring device by " film positive photoresist is born " principle.The min of 200 mA constant currents electrophoresis 40 is used during transferring film, by sample
Product protein delivery is on pvdf membrane.
Pvdf membrane is placed in 5 % skimmed milk powers and closes 3 h by transferring film after terminating.
Add DJ-1 antibody（Rabbit source, 1：500 dilutions）, 4 DEG C of overnight incubations.Rinsed, 10 min × 3, added peppery with TBST
The goat anti-rabbit igg of root peroxidase labelling（1：2000 dilutions）, 37 DEG C of 60 min of incubation.Rinsed again with TBST, 10 min
×3。
ECL luminescent solutions are added, react 5 min, gel imaging system imaging analysis.
2.2 experiment in vitro
2.2.1 the preparation of sperm suspension：Mouse bilateral epididymal is taken, is placed in the physiological saline of 37 DEG C of pre-temperatures, washes away hair
And blood, epididymis is put into the EP pipes of 1.5 ml physiological saline, eye scissors shreds, after sperm is free, by supernatant
Filtered with 4 layers of lens wiping paper, tally counts, and cell concentration is adjusted to 2 × 107/ ml, 37 DEG C standby.
2.2.2 eosin stains detect living spermatozoa percentage：0.5 g Eosin Ys are weighed, add 100 ml physiological saline, it is fully molten
Solution, filtering.Sperm suspension is equally divided into 4 groups, control group adds physiological saline, and the basic, normal, high concentration group of protocatechualdehyde adds former
Catechu aldehyde, it is respectively 0.01,0.1,1 mg/ml to make its concentration, and 37 DEG C of water-baths are incubated 0.5 h.Take processing after sperm suspension with
Each 1 drop of Yihong solution, mixes, is added dropwise on slide, is capped slide, place 30 ~ 60 s, light microscopic 400 × observation, Necrospermia head
Portion is colored pinkiness, and sperm living is not colored.200 sperms are counted, calculate living spermatozoa percentage.
2.2.3 sperm hypotonic expansion（hypo-osmotic swelling, HOS）The integrality of experiment detection sperm membrane：
0.3625 g sodium citrates are weighed, 0.675 g fructose adds pure water to be configured to ionic strength 0.15, osmotic pressure 150 to 50 ml
MOsm hypotonic medium.Sperm suspension is equally divided into 4 groups, control group adds physiological saline, the basic, normal, high concentration group of protocatechualdehyde
Protocatechualdehyde is added, makes its concentration be respectively 0.01,0.1,1 mg/ml, every group of ml of addition hypotonic medium 0.8,37 DEG C of water-baths 0.5
h.Sediment is drawn after water-bath to drop on cleaning slide, is capped slide humidity strip mounting, light microscopic 400 × observation, sperm tail shape
Shape change for swelling sperm, the afterbody swelling sperm count counted in 200 sperms is overall expansion rate.
2.3 data processing
Experimental data with± s is represented, is carried out statistical test with ANOVA, is compared P between group<0.05 is considered as difference
It is statistically significant.
Experimental result is with discussing
Influence of 3.1 protocatechualdehydes to mouse testis index, epididymis index and Maturation of sperm nucleus
Model group mouse testis index, epididymis index substantially reduce（P＜ 0.01）, and protocatechualdehyde can substantially increase mouse
Testis index and epididymis index（P＜ 0.05）.In addition model group mouse sperm Nuclear maturity degree is remarkably decreased（P＜ 0.01）, it is former
Catechu aldehyde can significantly improve mouse sperm Nuclear maturity degree（P＜ 0.01）, the results detailed in Table 1.
Testis is spermatogenetic vitals, and epididymis is the place of spermioteleosis, mouse after intraperitoneal injection of cyclophosphamide
Testis index and epididymis index reduce, and illustrate that mouse testis and epididymis sustain damage, and prompt mouse sperm generating process impaired, smart
Sub- maturity reduces；Mouse testis and epididymis index increase after protocatechualdehyde administration, illustrate that protocatechualdehyde can promote impaired testis
The reparation of ball and epididymis.
In spermatogenesis, the albumen that sperm nucleus DNA is combined can undergo the natural maturity mistake from histone to nucleoprotamine
Journey, this crosses referred to as male sterility, and this group of type conversion has important physiological significance.The conversion of nucleoprotein group type is different
Often, then immature sperm increases, and can produce substantial amounts of oxygen radical, destroys sperm membrane, changes sperm membrane permeability, so as to draw
Dead sperm is sent out, and then influences male fertility, will also result in the embryonic loss become pregnant.Aniline blue Coloration experiment shows, model
Group mouse sperm Nuclear maturity degree substantially reduces, and prompts endoxan that male sterility can be caused abnormal, and then may
Make the reduction of mouse fertility；And protocatechualdehyde can improve mouse sperm Nuclear maturity degree, protocatechualdehyde is prompted to improve male mice
Fertility.
3.2 protocatechualdehydes are on the morphologic influence of mouse testis
The histology of mouse testis（Reference picture 1）It has been shown that, model group mouse seminiferous tubule epithelium is thinning, lumen diameter
Increase, androgone arrangement disorder, the androgone number of plies are reduced, and oligozoospermia in tube chamber, seminiferous tubule gap substantially increases；It is former
Catechu aldehyde high dose group mouse testis form is similar to normal group, is arranged between each seminiferous tubule closely, thin on seminiferous tubule production of sperm
Born of the same parents' number of plies showed increased, each phase androgone marshalling is closely orderly, the visible more sperm of tube chamber, prompts protocatechualdehyde energy
Improve and recover seminaferous epithelium damage, seminiferous tubule structure is recovered normal, so that spermatogenesis recovers normal.
Fig. 1 is the influence for the Testis Morphology that protocatechualdehyde causes chmice in male sterile to endoxan（×400）, in figure：
a:Normal group, b:Model group, c:Protocatechualdehyde low dose group（10mg/kg）, d:Protocatechualdehyde high dose group（40mg/kg）.
Influence of 3.3 protocatechualdehydes to mouse testis tissue SOD activity
Compared with normal group, model group mouse testis tissue SOD activity is obvious to be reduced（P＜ 0.01）, the high agent of protocatechualdehyde
The activity of Liang Zu mouse testis tissue SOD dramatically increases（P＜ 0.05, reference picture 2）.
Fig. 2 is the influence that protocatechualdehyde causes chmice in male sterile testis tissue SOD activity to endoxan（± S, n=
10）, in figure：Compared with Normal group,## P<0.01；Compared with model group,* P<0.05。
There is research to think, the mechanism that endoxan causes spermatogenesis to damage is probably endoxan through hepatomicrosome P450
Enzyme system metabolism generates poisonous electrophilic material, and these electrophilic products cause radicals scavenging enzymatic activity to decline, cause freedom
Base is accumulated, and influences the activity of steroid hormone synzyme, and peroxidatic reaction of lipid occurs, and destroys biofilm structure, causes
Damage of Germ Cells, sperm microenvironment is set to change.SOD is the oxygen such as cell clearance ultra-oxygen anion free radical and hydrogen peroxide
The enzyme-specific of free radical.The tissue SOD's activity of model group mouse testis nine is decreased obviously in this experiment, hints model group mouse testis
By oxidativestress damage, so as to influence spermatogenesis and sperm function.Protocatechualdehyde can improve mouse testis tissue SOD
Activity, reduce oxidative stress in testis tissue, so as to recover the spermatogenesis of mouse.
Influence of 3.4 protocatechualdehydes to mouse testis tissue DJ-1 protein expressions
Compared with normal group, model group mouse testis tissue DJ-1 expression is obvious to be reduced（P＜ 0.01）, protocatechualdehyde is low,
High dose group mouse testis tissue DJ-1 expression dramatically increases（P＜ 0.05, reference picture 3）.
Fig. 3 is the influence that protocatechualdehyde causes chmice in male sterile testis tissue DJ-1 expression to endoxan（± S, n=
3）, in figure：Compared with Normal group,## P<0.01；Compared with model group,* P<0.05,** P<0.01。
DJ-1 albumen has obvious anti-oxidation stress function, is reached in male reproductive system altimeter, in each phase life of testis
Spermatid and interstitial cell have expression.Lot of documents shows that DJ-1 albumen and buck infertility caused by reproduction toxin are close
Cut is closed, and also has document to prove that DJ-1 albumen is related to mankind's azoospermia.It is a discovery of the invention that endoxan causes mouse testis group
Knitting DJ-1 protein contents substantially reduces, and this can cause the anti-oxidation stress miopragia of DJ-1 albumen, and then make testis tissue oxygen
Change stress level increase, so as to influence spermatogenesis.And protocatechualdehyde can improve mouse testis tissue DJ-1 protein contents,
So as to resist testis tissue oxidative stress, spermatogenesis is set to recover normal, and then improve mouse fertility.
Influence of the 3.4 protocatechualdehyde incubated in vitro to living spermatozoa percentage
Physiological saline and sperm suspension（Control group）It is 21.58% ± 5.98% to be incubated living spermatozoa percentage after 0.5 h, 0.01,
0.1st, 1 mg/ml protocatechualdehydes and sperm suspension are incubated, and are remarkably improved living spermatozoa percentage, be the results are shown in Table 2.
Sperm survival is the basis of sperm perform function activity, and no matter in vivo or in vitro, sperm must survive one regularly
Between could enter hyperactivation state, the experience prefecundation physiological change such as capacitation.This studies have shown that protocatechualdehyde and mouse essence
After son is incubated, the Motility rate of mouse sperm is remarkably improved, prompts protocatechualdehyde to improve sperm function.
Influence of the 3.5 protocatechualdehyde incubated in vitro to sperm membrane integrity
Control group sperm overall expansion rate is 20.90% ± 3.38%, 0.01,0.1,1 mg/ml protocatechualdehydes and sperm suspension
It is incubated, is remarkably improved sperm overall expansion rate, the results are shown in Table 3.
Sperm tail is the locomotive organ of sperm, and its function quality directly influences the energy of sperm.HOS is measure sperm
The simple and easy method of uropatagium integrality and function.Sperm tail is more loose than head construction, therefore to hypotonic very sensitive, in hypotonic environment
Various types of swelling can be presented.Generation swelling is that sperm membrane normally shows, therefore clinically more film work(that sperm is evaluated with this
Energy.After this studies have shown that protocatechualdehyde is incubated with mouse sperm, the percentage of expansion sperm is remarkably improved, prompts former catechu
Aldehyde can protect sperm membrane integrity in vitro, improve sperm membrane function.
Protocatechualdehyde treats the advantages of male sterility and beneficial effect：
For male sterility, the medical treatment regime that clinic is commonly used at present has：Hormone therapy, including antiestrogenic, rush
Gonadal hormone, gonadotropin-releasing hormone (GRH), androgen, Aromatase inhibitor；Antioxidation Treatment, including conventional vitamin
E, Co-Q10 etc.；Other drugs, including carnitine etc..
（One）Hormone therapy：Antiestrogenic curative effect is not affirmed, is not suitable for long-term treatment；The effect of promoting sexual gland hormone, has
Limit, application are less；Gonadotropin-releasing hormone (GRH) is costly, and curative effect is not good enough, does not recommend typically；Androgen in treating is to pregnant
Rate of being pregnent simultaneously is not improved；Aromatase inhibitor can not improve semen quality, and expensive, therefore clinical less use.
As can be seen here, hormone therapy clinically and is of little use, and expensive；And protocatechualdehyde abundance, therefore price is relatively low
It is honest and clean, it can promote the use of.
（Two）Antioxidation Treatment：Vitamin E curative effect is clearer and more definite, and its mechanism of action is mainly anti-oxidation stress, and vitamin E is not
Water is dissolved in, edible oil is contained in preparation, treatment male sterility long-term use Vitamin E preparation may cause patient's fat intake
Increase, so as to trigger relevant disease, such as dyslipidemia；And protocatechualdehyde good water solubility, patient's fat intake will not be increased.
Co-Q10（400 mg/d, 6 months）Patient's semen quality can be improved, by drug price, about 12 yuan of patient's daily cost, improved
Semen quality（6 months）About 2160 yuan of expense；And protocatechualdehyde price is less expensive.The mechanism of action of vitamin E and Co-Q10 is
It is anti-oxidant, and protocatechualdehyde also has other mechanism of action in addition to can be to anti-oxidation stress（Increase testis DJ-1 protein contents）.
（Three）Other：For carnitine at present using wide, carnitine treatment male sterility common dose be 2-3g/d, is joined
Examine drug price, patient's daily cost 22-33 members, 1 course for the treatment of（3 months）Medical expense 1980-2970 members, 2 courses for the treatment of（6
Month）Medical expense 3960-5940 members, expense are of a relatively high；And protocatechualdehyde abundance, and it is few per consumption per day（300 mg/
d）, therefore medical expense is lower than carnitine.The mechanism of action of carnitine is mainly anti-oxidant, and protocatechualdehyde is except can be to anti-oxidant
Stress be outer, there are other mechanism of action（Increase testis DJ-1 protein contents）.
In summary, it is carnitine and vitamin E that clinical treatment male sterility, which applies more medicine, at present, and the two is led
The mechanism of action wanted is anti-oxidation stress, and protocatechualdehyde is in addition to anti-oxidative stress, moreover it is possible to improves testis DJ-1 albumen
Content, it is better than carnitine and vitamin E in treatment male sterility mechanism.It is therefore believed that protocatechualdehyde is clinically
Can independent medication treatment male sterility, can be also used in combination with carnitine and vitamin E.
The advantages of protocatechualdehyde extracorporeal treatment sperm and beneficial effect：
Currently used sperm nutrient solution has Ham ' s-F10, Earle ' s, HTF etc., and its main component is various inorganic
Salt and amino acid, do not add other compositions.Protocatechualdehyde is added in sperm nutrient solution, its anti-oxidation stress work can be played
With caused oxygen radical during removing dealing sperm, so as to protect sperm morphology and function, than sperm is used alone
Nutrient solution effect is more.In addition, protocatechualdehyde is cheap, the running cost of extracorporeal treatment sperm will not be increased, and use
It is convenient（It is directly added into nutrient solution）, will not substantially increase operating procedure and time.
Claims (1)
1. a kind of protocatechualdehyde is preparing the application in preventing and treating caused by cyclophosphamide male sterility disease drug, it is characterised in that：Will
Protocatechualdehyde is used for male sterility and Reproductive Damage caused by preventing and treating endoxan.
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Non-Patent Citations (3)
| Title |
|---|
| "丹参对丝裂霉素C诱发小鼠生殖细胞遗传损伤的防护作用研究";赵景春 等;《癌变.畸变.突变》;19980131;第10卷(第1期);39-42 * |
| "丹参提取液在精液体外处理中的作用研究";陆遥 等;《辽宁中医杂志》;20020731;第29卷(第7期);432-433 * |
| "丹参水提物预防糖皮质激素对大鼠睾丸损害的实验研究";周乐 等;《广西医学》;20130331;第35卷(第3期);275-277 * |
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