CN105251017B - NONRATT021972 siRNA is in the application for preparing diabetes complicated cardiac autonomic nervous disease medicament - Google Patents
NONRATT021972 siRNA is in the application for preparing diabetes complicated cardiac autonomic nervous disease medicament Download PDFInfo
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Abstract
Long non-coding ribonucleic acid NONRATT021972 siRNA is preparing the application in diabetes complicated cardiac autonomic nervous disease medicament.Germicidal efficacy can improve the cardiac function anomalous variation of diabetic model rats to long non-coding ribonucleic acid NONRATT021972 siRNA, reduce raised blood glucose, serum inflammatory factors of senile level, oxidative stress, the activity for increasing antioxidant.After the processing of long non-coding ribonucleic acid NONRATT021972 siRNA, inhibit the up-regulated expression of diabetic model rats Superior cervical sympathetic ganglia gap connection hemichannel albumen 1 (Pannexin-1) with can dramatically, inhibit 302 phosphorylation of insulin receptor substrate-1 (IRS-1) aberrant serine, improves the IRS1 expression of downward.It can also be in the application in the disease mediated drug of Pannexin-1 and IRS1 dysfunction.Long non-coding ribonucleic acid NONRATT021972 siRNA carries out above-mentioned disease prevention and cure with oral, injection, lozenge or other locally or systemically drug formulation drugs.
Description
Technical field
The present invention relates to diabetes complicated cardiovascular autonomic neuropathy medicinal usage invention fields.
Background technique
Diabetes (Diabetes Mellitus, DM) are one group of metabolic clinical syndromes, development and people with society
The illness rates of improvement of living standard diabetes increase year by year, reached 3%-7% in developed country's diabetes prevalence, become
It is only second to after cancer, AIDS, cardiovascular and cerebrovascular diseases the 4th and needs top-priority disease, it has also become the 5th, the world is dead main
Cause.Diabetes are divided into 1 type (insulin-dependent) and 2 types (non-insulin-dependent) diabetes.It is estimated that in global six people
Face just has a people to be in the danger for suffering from diabetic complication.The composition of China's diabetic population is accounted for based on diabetes B
90% or more of diabetic population, drastically influences people's health and social development.Diabetic autonomic neuropathy (Diabetic
Autonomic neuropathy, DAN) it is one group of syndrome caused by autonomic nervous function and (or) structural damage, mainly
Involve cardiovascular, gastrointestinal tract and urogenital system, there is onset concealment, be gradually in progress, can occur before symptom occurs, is very few
The Clinical symptoms voluntarily alleviated.Wherein, diabetic heart autonomic neuropathy (Diabetic cardiac autonomic
Neuropathy, DCAN) harm be particularly acute, silent myocardial ischemia, myocardial infarction and malignant arrhythmia can be caused very
To sudden cardiac death.Autonomic nerve includes sympathetic and parasympathetic nerve, is passed through from the sympathetic or parasympathetic nerve of maincenter downlink outer
All autonomic ganglias dominate internal organs such as heart etc. after changing neuron.The variation of heart rate and blood pressure is directly by parasympathetic system
System (sympathetic nerve and parasympathetic nerve) movable influence.
Pannexin-1 gene is the inserted by connexin family newcomer of discovery in 2000, research shows that Pannexin-1
Albumen can form hemichannel on cell membrane or form Gap junctions in iuntercellular, participate in a variety of important physiological and pathologicals
Process.Gap connection hemichannel (hemichannels) be Gap junctions subunit, in vertebrate body each
Six aggressiveness of cross-film that hemichannel is all made of Connexin or Pannexin albumen is constituted, two hemichannels of flanking cell
Docking forms hollow pipeline two-by-two, mediates the flowing of small-molecule substance, realize cell-tocell and substance quick exchange and
It links up.The abnormal activation of Pannexin-1 hemichannel, effect of extracellular ATP dramatically increase under pathologic condition, and act on purinoceptor,
The ATP release of ATP induction to activate Pannexin-1 hemichannel to mediate, and vicious circle is formed, cause extracellular a large amount of
The aggregation of ATP causes the abnormal flow of various ions, so as to cause the disorder of intracellular ion and the missing of energy, occurs thin
The swelling of born of the same parents and the collapse of cell membrane, to be related to the multiple functions disorder and disease of inflammation, immune system and nervous system.
IRS1 is the key protein in insulin signaling pathway, participates in the growth and metabolism of cell.The phosphorylation of IRS for
The transduction of insulin signaling pathway is critically important, and tyrosine site phosphorylation can make signal normal conduction, but IRS exception silk ammonia
The phosphorylation of acid often results in the degradation of IRS to disturb the conduction of insulin signaling, it may be possible to cause the weight of insulin resistance
The molecular mechanism wanted.There are a series of performances of diabetes B: the insulin resistance of surrounding tissue in the mouse for knocking out IRS1, high
Blood glucose and β cell secretory functional disturbance, β cell number are reduced.Have been reported that document IRS aberrant serine phosphorylation can mediate
Dorsal root ganglion neurons insulin resistance, and it is related to the pathogenesis of diabete peripheral herve pathology.
Numerous studies confirm, when diabetes high glucose and high fat toxicity, generating excessive free radical in vivo cannot be removed
Generate oxidative stress enhancing.This research experiment is the result shows that diabetes rat fasting blood-glucose and postprandial blood sugar apparent increase, serum
Oxidative stress index malonaldehyde (MDA) increases, glutathione peroxidase (GSH-PX) and total nitricoxide synthase (T-NOS)
Activity decline, prompting diabetes rat Superior cervical sympathetic ganglia, there are apparent oxidative stress.Oxidative stress can induce a large amount of promote
Scorching factor synthesis release, causes inflammatory reaction to influence the function of nerve cell and changes the microenvironment of nerve cell, participate in
Or accelerate the generation of diabetes and its cardiovascular autonomic neuropathy, development.Inflammatory cytokine such as tumor necrosis factor-alpha (TNF-
α) the activation of access, it is related with the generation of the chronic complicating diseases of diabetes including neuropathy.
The inhereditary material of the mankind and other higher eucaryotes only has minimum a part of coding protein, and is more than 97%
Transcription product is ribonucleic acid vdiverse in function (RNA) molecule, their functions without coding protein are referred to as non-coding ribose
Nucleic acid (noncoding RNA, ncRNA).Non-coding RNA adjusts gene expression with rna form.Non-coding RNA is roughly divided into 2
Class: " special RNA " and " rna regulation "." special RNA " generally belongs to constitutive expression, is cells survival and basic function
Required composition." rna regulation " can be divided into transcription regulator, the post-transcriptional control factor, RNA distribution regulatory factor, protein function
Regulatory factor etc.." rna regulation " includes miRNA (miRNA), small interference ribonucleic acid (siRNA), transcribed vacation
Gene, antisense RNA, core regulator etc..Small non-encoding ribonucleic acid (such as miRNA, siRNA), length can be divided into again by adjusting ncRNA
In 200nt to the long non-coding ribonucleic acid (Long noncoding RNA, LncRNA) for being more than 100kb.With more and more
Noncoding gene and its function are identified and disclose, though people gradually recognize function of the lncRNA without coding protein, and
Protein molecule is equally important, and even main functional molecular plays important and extensive in cell normal activities
Adjustment effect.The variation of research prompt lncRNA expression and the factor for influencing lncRNA change related with disease.We are real
It tests the screening of room SOLiD early period high throughput rat transcript profile database and molecular biology is verified and determines that neck sympathetic ganglion exists
Long non-coding ribonucleic acid NONRATT021972 [http://www.noncode.org], is submitted in diabetic model rats neck
Sense neuromere long non-coding ribonucleic acid NONRATT021972 expression obviously increases.This research is long by diabetic model rats
Non- encoding ribonucleic acid NONRATT021972 small interference ribonucleic acid (RNA) inhibits its expression, observes long non-coding ribonucleic acid
Effect and its regulatory mechanism of the NONRATT021972 siRNA in diabetes complicated cardiovascular autonomic neuropathy, this is to spy
The novel targets of the pathogenesis and prevention and treatment of seeking diabetic neuropathy are of great significance.
Summary of the invention
The first purpose of this invention is provide long non-coding ribonucleic acid NONRATT021972 siRNA
One new application, i.e. long non-coding ribonucleic acid NONRATT021972 siRNA treat diabetes complicated heart certainly in preparation
Application in main neurological disease drug.
Second object of the present invention is provide long non-coding ribonucleic acid NONRATT021972 siRNA
Two new applications, i.e. long non-coding ribonucleic acid NONRATT021972 siRNA treat sympathetic nerve related disease in preparation
Application in drug.
Third object of the present invention is provide long non-coding ribonucleic acid NONRATT021972 siRNA
Three new applications, i.e. long non-coding ribonucleic acid NONRATT021972 siRNA are preparing general connection albumen -1
(Pannexin-1) application in the drug for the related disease that hemichannel mediates.
Fourth object of the present invention is provide long non-coding ribonucleic acid NONRATT021972 siRNA
Three new applications, i.e. long non-coding ribonucleic acid NONRATT021972 siRNA are in preparation insulin receptor substrate-1
(IRS1) application in the drug for the related disease that dysfunction mediates.
The present invention observes the small interference of long non-coding ribonucleic acid NONRATT021972 by diabetes B rat model
Effect and its regulatory mechanism after RNA processing in the concurrent cardiovascular autonomic neuropathy of diabetes B, are long non-coding ribonucleic acid
Prevention and treatment of the NONRATT021972 siRNA for diabetes complicated concurrent cardiovascular autonomic neuropathy provide help.
The present invention is expressed by establishing diabetes B rat model, and to the Superior cervical sympathetic ganglia height filtered out early period
Long non-coding ribonucleic acid NONRATT021972 carries out siRNA, using Microarrays long non-coding ribose core
Cause the change of Superior cervical sympathetic ganglia cell function related gene after sour NONRATT021972 siRNA processing, further
It is small to long non-coding ribonucleic acid NONRATT021972 dry by Double immunofluorescence, Real-time PCR and Western blotting
It disturbs and changes most apparent gene after RNA and verified, observe blood after the small interference of long non-coding ribonucleic acid NONRATT021972
Sugar, serum interleukin-6 (IL-6), tumor necrosis factor (TNF-α), norepinephrine (NE) and oxidative stress refer to
The variation for marking malonaldehyde (MDA), Glutathione peroxidase (GSH-PX) and total nitricoxide synthase (T-NOS), in conjunction with length
Rat blood pressure after non-encoding ribonucleic acid NONRATT021972 siRNA, heart rate, Electrocardiograph index, heart rate variability and dynamic
The difference that the hearts autonomic functions such as arteries and veins blood pressure pressure sensibility are shown evaluates long non-coding ribonucleic acid NONRATT021972
Effect and possible mechanism of the siRNA in the damage of Superior cervical sympathetic ganglia mediate diabetic autonomic nervous function.Pass through
This experimental study is to further appreciating that long non-coding ribonucleic acid NONRATT021972 siRNA in treating diabetes
Effect and its novel targets that may act on, and the prevention and treatment for diabetes and its complication provides new experimental basis.
That researches show that diabetic model rats blood glucose is significantly raised by the present invention, and there are apparent oxidative stress,
The inflammatory factor release that reconciles in Pannexin-1 expression increases, and the expression of IRS1 aberrant serine phosphorylation and IRS1 are lowered, the heart
Dirty autonomic dysfunction；After long non-coding ribonucleic acid NONRATT021972 siRNA, reduce rat blood sugar and
Oxidative stress inhibits the release of the upper reconciliation inflammatory factor of diabetes Superior cervical sympathetic ganglia Pannexin-1 expression, drop
Low diabetes Superior cervical sympathetic ganglia IRS1 aberrant serine phosphorylation improves IRS1 expression and lowers and promote sympathetic nerve on neck
Ganglion cell's growth generates protective effect to the cardiovascular autonomic nervous function that diabetes are damaged.
Long non-coding ribonucleic acid NONRATT021972 siRNA is in prevention and treatment diabetes and the effect machine of complication
Reason is related to: inhibiting to cause Superior cervical sympathetic ganglia/sympathetic PC12 cell long non-coding ribonucleic acid with high glucose and high fat toxicity
NONRATT021972 high expresses relevant inserted by connexin Pannexin-1 hemichannel, insulin receptor substrate-1
The expression of (insulin receptor substrate-1, IRS-1) generates diabetes complicated disease and other related diseases
Preventive and therapeutic effect.
To better understand the essence of the present invention, below with the small interference of long non-coding ribonucleic acid NONRATT021972
Effect of the RNA to diabetes B rat model sympathetic nerve, painstaking effort dysfunction, and to the gap on Superior cervical sympathetic ganglia
Albumen Pannexin-1 hemichannel, the influence experiment of insulin receptor substrate-1 expression and correlated results are connected to prove to grow non-volume
The purposes of code ribonucleic acid NONRATT021972 siRNA.
Detailed description of the invention
Fig. 1 is the variation diagram that the present invention tests each period each group rat blood serum IL-6.Wherein Fig. 1 (a) is experiment the 5th week
End, Fig. 1 (b) are the 8th weekend of experiment.Experimental group: Normal group, diabetic model group, diabetes model+long non-coding core
Ribosomal ribonucleic acid NONRATT021972 siRNA group and diabetes model NCsi group；
Fig. 2 is the variation diagram of the present invention the 8th weekend each group rat blood serum tumor necrosis factor-alpha (TNF-α) of experiment.Experiment
Grouping: Normal group, diabetic model group, diabetes model+small interference of long non-coding ribonucleic acid NONRATT021972
RNA group and diabetes model+random ordering siRNA negative control group；Wherein**P < 0.01 is indicated and normal group is compared,##p<
0.01 indicates compared with diabetic model group.
Fig. 3 is the variation diagram of the present invention the 8th weekend each group rat blood serum norepinephrine (NE) of experiment.Experimental group:
Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid NONRATT021972 siRNA group and
Negative (NCsi) control group of diabetes model+random ordering siRNA；Wherein**P < 0.01 is indicated and normal group is compared,##p<
0.01 indicates compared with diabetic model group.
Fig. 4 is the general connection albumen -1 (Pannexin-1) of Superior cervical sympathetic ganglia cell of the present invention and neuronal marker
(NeuN) the double mark result figures of antibody mediated immunity fluorescence.Diabetic model group P of Rats annexin-1 expression is apparently higher than normal control
Group, the Pannexin-1 of rat model is expressed and is reduced after the processing of long non-coding ribonucleic acid NONRATT021972 siRNA.
Experimental group: Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid NONRATT021972 are small dry
Disturb RNA group and diabetes model+random ordering siRNA negative control group；
Fig. 5 is 1 (IRS-1) IRS1 receptor of Superior cervical sympathetic ganglia cell insulin receptor substrate-1 of the present invention and neuron
The double mark result figures of marker (NeuN) antibody mediated immunity fluorescence.Diabetic model group rat Superior cervical sympathetic ganglia IRS1 expression of receptor
Substantially less than Normal group, rat model neck is submitted after the processing of long non-coding ribonucleic acid NONRATT021972 siRNA
Feel neuromere IRS1 expression to increase.Experimental group: Normal group, diabetic model group, diabetes model+long non-coding ribose
Nucleic acid NONRATT021972 siRNA group and diabetes model+random ordering siRNA negative control group；
Fig. 6 is that real-time quantitative PCR of the present invention detects Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972
Expression variation diagram.Diabetic model group rat Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972 expression
It is apparently higher than Normal group, after the processing of long non-coding ribonucleic acid NONRATT021972 siRNA on the neck of rat model
Sympathetic ganglion long non-coding ribonucleic acid NONRATT021972 expression reduces.Experimental group: Normal group, diabetes mould
Type group, diabetes model+long non-coding ribonucleic acid NONRATT021972 siRNA group and diabetic model group+random ordering
SiRNA negative control group；Wherein**P < 0.01 is indicated and normal group is compared,##P < 0.01 is indicated and diabetic model group ratio
Compared with.
Fig. 7 is the mRNA expression variation diagram that real-time quantitative PCR of the present invention detects Superior cervical sympathetic ganglia Pannexin-1.Sugar
The mRNA expression of urine disease model group rat Superior cervical sympathetic ganglia Pannexin-1 is apparently higher than Normal group, long non-coding core
The mRNA table of the Superior cervical sympathetic ganglia Pannexin-1 of rat model after the processing of ribosomal ribonucleic acid NONRATT021972 siRNA
Up to reduction.Experimental group: Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid
NONRATT021972 siRNA group and diabetes model+random ordering siRNA negative control group；Wherein**The table of p < 0.01
Show and compare with normal group,##P < 0.01 is indicated compared with diabetic model group.
Fig. 8 is the mRNA expression variation diagram that real-time quantitative PCR of the present invention detects Superior cervical sympathetic ganglia IRS1.Diabetes mould
The mRNA expression of type group rat Superior cervical sympathetic ganglia IRS1 is apparently higher than Normal group, long non-coding ribonucleic acid
The mRNA of the Superior cervical sympathetic ganglia IRS1 of rat model is expressed and is reduced after the processing of NONRATT021972 siRNA.Experiment point
Group: Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid NONRATT021972 siRNA
Group and diabetes model+random ordering siRNA negative control group；Wherein**P < 0.01 is indicated and normal group is compared,##p<0.01
It indicates compared with diabetic model group.
Fig. 9 is that western blotting technique of the present invention detects Superior cervical sympathetic ganglia Pannexin-1 protein expression variation diagram.Sugar
Urine disease model group rat Superior cervical sympathetic ganglia Pannexin-1 protein expression is apparently higher than Normal group, long non-coding ribose
The Superior cervical sympathetic ganglia Pannexin-1 protein expression drop of rat model after the processing of nucleic acid NONRATT021972 siRNA
It is low.Experimental group: Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid NONRATT021972
SiRNA group and diabetes model+random ordering siRNA negative control group；Fig. 9 (a) is protein blot experiment result figure,
Fig. 9 (b) compares histogram for analysis of experimental data, wherein**P < 0.01 is indicated and normal group is compared,##P < 0.01 is indicated and glycosuria
Disease model group compares.
Figure 10 is that western blotting technique of the present invention detects Superior cervical sympathetic ganglia IRS1 and p-IRS ser302 protein expression
Variation diagram.Diabetic model group rat Superior cervical sympathetic ganglia IRS1 protein expression is significantly lower than Normal group, long non-coding
The Superior cervical sympathetic ganglia IRS1 protein expression of rat model increases after the processing of ribonucleic acid NONRATT021972 siRNA.
Diabetic model group rat Superior cervical sympathetic ganglia p-IRS ser302 protein expression is apparently higher than Normal group, long non-coding
The Superior cervical sympathetic ganglia p-IRS ser302 albumen of rat model after the processing of ribonucleic acid NONRATT021972 siRNA
Expression reduces.Experimental group: Normal group, diabetic model group, diabetes model+long non-coding ribonucleic acid
NONRATT021972 siRNA group and diabetes model+random ordering siRNA negative control group；Figure 10 (a) is albumen
Blot experiment result figure, Figure 10 (b) compare histogram for analysis of experimental data, wherein**P < 0.01 is indicated and normal group is compared,
In**P < 0.01 is indicated and normal group is compared,##P < 0.01 is indicated compared with diabetic model group.
Specific embodiment
Below with reference to embodiment and compares attached drawing invention is further described in detail.
Embodiment 1.
With method well-known in the art, it is made suitable for the oral of diabetes complicated cardiac autonomic nervous disease treatment
Or the long non-coding ribonucleic acid NONRATT021972 siRNA preparation of injection.
Embodiment 2.
With method well-known in the art, the oral or injection for being applicable to sympathetic nerve treating correlative diseases is made
Long non-coding ribonucleic acid NONRATT021972 siRNA preparation.
Embodiment 3.
With method well-known in the art, it is made and is applicable to the disease mediated treatment of Pannexin-1 dysfunction
Oral or injection long non-coding ribonucleic acid NONRATT021972 siRNA preparation.
Embodiment 4.
With method well-known in the art, be made be applicable to the oral of the disease mediated treatment of IRS1 dysfunction or
The long non-coding ribonucleic acid NONRATT021972 siRNA preparation of injection.
In short, long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid with oral, injection, lozenge or its
Locally or systemically drug formulation drug carries out above-mentioned disease prevention and cure for it.
To better understand the essence of the present invention, below with the small interference of long non-coding ribonucleic acid NONRATT021972
Ribonucleic acid proves long non-coding to the experiment of the sympathetic autonomic nerve disease treatment Effect study of the diabetes complicated heart and result
The purposes of ribonucleic acid NONRATT021972 small interference ribonucleic acid.
One, material and method
1. establishing diabetes B rat model.
SD male rat (weight 180g or so), sub-cage rearing, every cage raising 7,20-25 DEG C of temperature, relative humidity
40%-70%, light and shade replaces 12h/12h round the clock, and free water cuts tail blood sampling and surveys fasting blood-glucose and 2 hours postprandial after adapting to 1 week
Blood glucose.Rat is randomly divided into Normal group (15) and model treatment group (60), Normal group is fed commonly always
Feed (is provided) by Laboratory Animal Science portion of medical college of University Of Nanchang, and model group feeds high glucose and high fat feed (basal feed
66.5%, sucrose 20%, lard 10%, cholesterol 2.5%, sodium taurocholate 1% adds water to do and is placed on constant temperature blast drying oven into strips
In 80 DEG C it is 3 hours dry roasting).5th weekend (after i.e. model group is fed high glucose and high fat feed 4 weeks), all rats cut tail blood sampling and survey on an empty stomach
Blood glucose and postprandial 2 hours blood glucose.Streptozotocin (STZ) 30mg/kg is injected intraperitoneally (before use by STZ in model group rats on an empty stomach
In the 0.1mol/L sodium citrate-citric acid buffer that the pH for being dissolved in the pre-cooling of 4 DEG C of refrigerators is 4.2, it is configured to the STZ of 2.5g/L
Solution).The modeling group individual of 6th weekend, fasting blood-glucose < 7.8mmol/L and postprandial 2 hours blood glucose < 11.1mmol/L are empty again
Abdomen intraperitoneal injection STZ 30mg/kg is primary, and the 7th weekend surveyed fasting blood-glucose and postprandial 2 hours blood glucose, with fasting blood-glucose >
2 hours blood glucose > 11.1mmol/L of 7.8mmol/L or postprandial determine Glycemia Decline success.
2. the experiment of diabetes rat long non-coding ribonucleic acid NONRATT021972 siRNA and animal packet.
Long non-coding ribonucleic acid NONRATT021972 siRNA (siRNA) sequence is by Invitrogen
(Carlsbad, CA) company provides, target sequence 5 '-GAATGTTGGTCATATCAAA-3 '；Negative control scrambled
SiRNA (out-of-order small interference ribonucleic acid, NCsi) is purchased to Invitrogen (Carlsbad, CA) company.Body transfection reagent by
EntransterTMCompany provides.According to EntransterTMBody transfect specification, experiment the 7th week foot couple Glycemia Decline at
The rat of function carries out long non-coding ribonucleic acid NONRATT021972-siRNA in the small interference experiment of body.320 μ L siRNA are (long
Non- encoding ribonucleic acid NONRATT021972si) or negative control siRNA (NCsi) solution diabetes are injected by sublingual vein
In rat model body, rat is randomly divided into Normal group (control), diabetes rat model group (DM), diabetes rat
Model long non-coding ribonucleic acid NONRATT021972 siRNA processing group (long non-coding ribonucleic acid
NONRATT021972si) and diabetes rat model siRNA control treatment group (NCsi), wherein Normal group
(control) and 320 μ L of diabetes rat model group (DM) sublingual vein injecting normal saline.During experiment, rat freely drinks
Water, remaining each group continues to feed 1 week in addition to Normal group with high glucose and high fat feed.Respectively at the 5th weekend and the 8th weekend,
Measure each group rat index of correlation and blood sampling, collect specimen after the 8th weekend tested.
3. drug and reagent.
Transfection reagent (Engreen in animal bodyTMCompany), long non-coding ribonucleic acid NONRATT021972-siRNA,
Scrambled siRNA (Invitrogen company), β-actin, long non-coding ribonucleic acid NONRATT021972,
Pannexin-1 and IRS1 primer (Generay Biotech company), rabbit-anti Pannexin-1 antibody (Abcam company), rabbit-anti
IRS1 antibody (Upstate Biotechnology company), rabbit-anti p-IRS1ser302 antibody (Santa cruz company), chain urea
Assistant rhzomorph (Sigma company), rat tumor necrosis factor-alpha quantify elisa kit for detecting, and (Wuhan Yi Lai Rett biotechnology has
Limit company), Rat Interleukin -6 quantify elisa kit for detecting (Wuhan Yi Lai Rett Biotechnology Co., Ltd), rat
Norepinephrine elisa kit for detecting (Shanghai Sen Xiong Scientific and Technical Industry Co., Ltd).
4. key instrument.
The full vigor type blood glucose meter of Luo Kang (German Roche Holding Ag)；Softron intelligence non-invasive blood pressure measuring-mouse note surveys instrument (Gene&
I company)；Antiseptic gauze, towel, cotton swab etc., the tincture of iodine and 75% alcohol, surgical kit: scissors, ophthalmology tip-curved forceps, vessel forceps,
Silk thread, pincers etc..
5. the measurement of rat blood sugar.
Each group rat cuts tail blood sampling respectively at adaptation 1 week, the 5th weekend and the 8th weekend, using the full vigor type blood glucose meter of Luo Kang
Fasting blood-glucose and fasting 12h after measuring fasting 12h and by the postprandial blood sugar after glucose 2g/kg dosage stomach-filling 2h.
6. rat blood pressure, heart rate measurement.
Each group rat uses BP-2006A intelligence non-invasive blood pressure measuring to monitor respectively at adaptation 1 week, the 5th weekend and the 8th weekend
Normal group (control), diabetes rat model group (DM), diabetes rat model long non-coding ribonucleic acid
NONRATT021972 siRNA processing group (long non-coding ribonucleic acid NONRATT021972si) and diabetes rat model
The variation of siRNA control treatment group (NCsi) rat blood pressure and heart rate.In quietly monitoring room, by the rat of waking state
It being put into the mouse bag of the net containing mouse, tail is placed in mouse bag outside, and mouse bag temperature control is at 37 DEG C, after rat is quiet, pressurization induction
Device covers at the one third of rat-tail root, starts noninvasive intelligent sphygmomanometer, and into monitoring state, clicking start button can automatic measurement
The variation of each group rat blood pressure and heart rate.Same rat duplicate measurements blood pressure and heart rate three times in the same time, takes it average
Value, operation is completed by same people every time.
7. the measurement of Rat Ecg and heart rate variability (heart rate variability, HRV) index.
10% chloraldurate (0.3ml/100g) is injected intraperitoneally to be anaesthetized.Rat takes dorsal position after anesthesia, is fixed on hand
Art platform, ECG electrode is fixed on double upper limbs and right lower extremity is subcutaneous, and shirtfront preserved skin, disinfection use Medlab biological signal collecting
The electrocardiogram of processing system record standard II lead.The electrocardiosignal of acquisition heart rate variability analysis software is analyzed, is used
Journey 5min frequency domain analysis in short-term takes 5min electrocardiogram to make HRV analysis.The related parameter of HRV frequency domain analysis:
Total frequency spectrum (Total power, TP): being that power frequency density is bent for the general power of NN interphase variation in 24 hours
Integrated value of the line function within the scope of 0Hz-0.4Hz, that is, power spectrum density curve within the scope of 0Hz-0.4Hz with cross
Area folded by axis, unit ms2。
Very low frequencies (Very low frequency, VLF): being the very low frequencies component curve that power spectral density plot resolves into
The integrated value of (centre frequency is located at 0.003Hz-0.04Hz), that is, area folded by very low frequencies component curve and horizontal axis, it is single
Position is ms2。
Low frequency (Low frequency, LF): for the product of low frequency component curve (centre frequency is located at 0.04Hz-0.15Hz)
Score value, that is, area folded by low frequency component curve and horizontal axis, unit ms2。
High frequency (High frequency, HF): for the product of high fdrequency component curve (centre frequency is located at 0.15Hz-0.4Hz)
Score value, that is, area folded by high fdrequency component curve and horizontal axis, unit ms2。
Low frequency/high frequency (LF/HF): the ratio of low frequency and high frequency power illustrates sympathetic tone and vagal tone
Balance.
7. arterial pressure Baroreflex Sensitivity (baroreflex sensitivity, BRS).
It is injected intravenously neo-synephrine (5mg/kg) with Harverd infusion pump, increasing blood pressure, (stimulation arterial pressure is experienced
Device), then reflectivity slows down heart rate.Continuous monitoring each group rat blood pressure and electrocardiogram R -- R interval (cardiac cycle) variation, with artery
It is abscissa that systolic blood pressure, which increases variation, and following the extended R -- R interval of reflectivity of blood pressure is ordinate, is carried out to the two
Linear regression analysis.General in linear relation between the two, the slope of regression straight line is arterial baroreceptor reflex sensitivity
Property.When its meaning is arterial pressure every raisings 0.13kPa (1mmHg), the extended millisecond number of corresponding reflexive R -- R interval, with ms/
MmHg is indicated.Slope is big, prompts vagus reflex enhancing；Slope is small, and vagus reflex is prompted to weaken, and reflects sympathetic
Neural activity enhancing.
8. enzyme-linked immunosorbent assay (ELISA) method is tested.
1) sample prepares: the 5th weekend and the 8th weekend, collects serum, -80 DEG C of preservations；
2) it tests first 20 minutes and takes out kit from refrigerator, with balance to room temperature (20-25 DEG C)；
3) required amount of lath is taken out, remaining sealing puts back to 4 DEG C；
4) establish standard curve: if 3 multiple holes 100 μ l of sample diluting liquid is added, the first hole adds in 8 hole of gauge orifice in every hole
100 μ l of standard items is sucked out 100 μ l with sample injector after mixing, moves to the second hole.It opposes repeatedly and is diluted to seven apertures in the human head again, most
Afterwards, 100 μ l are sucked out from seven apertures in the human head to discard, being allowed to volume is 100 μ l.Octal is blank control；
5) be loaded: 100 μ l of sample to be tested is added in every hole in sample wells to be measured；
6) reaction plate is set 37 DEG C 120 minutes；
7) board-washing: sufficiently being washed reaction plate 4-6 times with cleaning solution, is printed on filter paper dry；
8) 50 μ l of first antibody working solution is added in every hole；
9) reaction plate is mixed well 37 DEG C of postposition 60 minutes；
10) board-washing: sufficiently being washed reaction plate 4-6 times with cleaning solution, is printed on filter paper dry；
11) the enzyme 100 μ l of labeling antibody working solution in every hole；
12) reaction plate is set 37 DEG C 60 minutes；
13) board-washing: sufficiently being washed reaction plate 4-6 times with cleaning solution, is printed on filter paper dry；
14) 100 μ l of substrate solution is added in every hole, sets 37 DEG C of dark places and reacts 5 minutes；
15) every hole is added 50 μ l terminate liquids and mixes；
16) side light absorption value (should as early as possible, it is higher that overlong time may cause background) at 450nm.
9. the measurement of oxidative stress index.
1) malonaldehyde (MDA) measures: to there is lid centrifuge tube (5ml) to be separately added into 0.05ml standard items (10nmol/ml), nothing
Water-ethanol and sample mix later plus equivalent reagent one；Two 1.5ml of reagent adding, three 0.5ml of reagent, swirl mixing device mix,
95 DEG C water-bath 40 minutes, flowing water is cooling after taking-up, then 3500 revs/min, is centrifuged 10 minutes.It takes at supernatant l ml, 532nm, 1cm
Optical path, distilled water zeroing, surveys each pipe absorbance value.Calculation formula: MDA content (nmol/ml)=(measurement pipe absorbance-measurement
Blank tube absorbance) dilution times before/(standard pipe absorbance-standard blank tube absorbance) × standard concentration × test sample
Number.
2) total nitricoxide synthase (T-NOS) vitality test: measurement pipe plus distilled water 0.1ml and sample 0.03ml, control
Pipe plus distilled water 0.13ml, shake up；One 0.2ml of reagent, two 0.01ml of reagent, reagent three is added in measurement pipe and control tube
0.1ml, swirl mixing device mix well, and set 37 DEG C of waters bath with thermostatic control 15 minutes；Four 0.1ml of reagent kit and reagent five is added in every pipe
2ml, swirl mixing device mix well, and at wavelength 530nm, each pipe absorbance value is surveyed in 1cm optical path, distilled water zeroing.It calculates public
Formula: T-NOS vigor (U/m1)=(measurement pipe absorbance-blank tube absorbance)/colour generation object nanomole extinction coefficient × reactant
It is total volume/sampling amount × 1/ colorimetric optical path × reaction time × 1/1000, colour generation object nanomole extinction coefficient takes 38.3 × 10-6。
3) glutathione peroxidase (GSH-PX) determination of activity: measurement pipe and control tube distinguish 1mmol/L reduced form
Glutathione (GSH) 0.2ml, reagent one 0.1ml, 37 DEG C pre-temperature 5 minutes；Sample/distilled water 0.2ml is added, 37 DEG C accurate anti-
It answers 5 minutes；Two 2ml of reagent, is mixed well with swirl mixing device, 4000 revs/min, is centrifuged 10 minutes, takes supernatant 1ml to do colour developing anti-
It answers.Blank tube adds reduced glutathione standard items solvent application liquid 1ml, standard pipe to add 20 μm of ol/L reduced glutathione marks
Quasi- liquid 1ml, control tube and measurement Guan Jun add clear liquid 1ml, and three 1ml of reagent, four 0.25ml of reagent, reagent is added in above four pipe
Five 0.05ml mix, are placed at room temperature for 15 minutes, and at wavelength 412nm, 1cm optical path cuvette, distilled water zeroing is surveyed each pipe and inhaled
Shading value.Calculation formula: GSH-PX vigor (U/m1)=(control tube absorbance-measurement pipe absorbance)/(standard pipe absorbance-
Blank tube absorbance) extension rate before × standard concentration × extension rate × test sample.Standard pipe concentration takes 20 μm of ol/L,
Extension rate is 6.
10. immunohistochemistry.
1) take out rat Superior cervical sympathetic ganglia frozen section restore room temperature after, wash 3 times with 0.1M PBS, 5min/ times,
4% paraformaldehyde room temperature pre-fixes 15min；
2) 0.1M PBS is washed 3 times, 5min/ times；37 DEG C of incubation 1h of lowlenthal serum confining liquid；
3) the diluted gap rabbit source 1:100 is added and connects hemichannel albumen 1 (Pannexin-1), insulin receptor substrate
1 (IRS1) primary antibody and the diluted NeuN primary antibody of mouse source 1:100,37 DEG C of water-baths are incubated for 2h；
4) 0.1M PBS is washed 3 times, 5min/ times；
5) the TRITC label 1 of FITC label the 1:200 diluted secondary antibody and sheep anti-Mouse source in goat-anti rabbit source is added:
200 diluted secondary antibodies are protected from light 37 DEG C of water-baths and are incubated for 45min, are protected from light；
6) 0.1M PBS is washed 3 times, 5min/ times；
7) fluorescence mountant mounting, is protected from light.It takes pictures under fluorescence microscope, analyzes result.
Real-time quantitative polymerase chain reaction 11. (Real-time PCR) technology.
1) extraction of total serum IgE.
A) at the end of the experiment of the 8th weekend, taking-up Superior cervical sympathetic ganglia is put into be cleaned in the processed PBS of DEPC；
B) it is transferred to respectively again with the processed homogenizer of DEPC, the Trizol that 1ml is added is fully ground；
C) homogenised sample is placed at room temperature for 5min, is kept completely separate nucleic acid-protein compound；
D) after every pipe addition 0.2ml chloroform mixes well, ice bath 15 minutes；
E) 4 DEG C of centrifugation 12000g × 15min, it is seen that the liquid in pipe be divided into (colourless liquid phase contain total serum IgE), in (white
Emulsus contains protein) and under (red containing DNA) three-phase；
F) upper phase is moved into the EP pipe of another 1.5ml through DEPC water process, adds isometric isopropanol, overturned mixed
It is even to set -20 DEG C about 20 minutes for several times, precipitate RNA；
G) 4 DEG C of centrifugation 12000g × 15min abandon supernatant, the visible a little white depositions of tube bottom (for RNA)；
H) every pipe adds 75% ethyl alcohol 1ml, sufficiently washing RNA precipitate, 4 DEG C of centrifugation 10000g × 15min to abandon supernatant × 2 time；
I) supernatant is abandoned, 10 minutes is stood, is allowed to drying, adds 50 μ l to be resuspended without RNase water and precipitates, 20 μ l RNA is taken to be used for
Quantitative and purity detecting, remaining -20 DEG C of preservations.
2) identification of total serum IgE.
A) it spectrophotometer detection (RNA concentration, purity testing): is returned to zero with 0.1%DEPC water to colour comparatour, takes 2 μ l
RNA sample surveys OD 260nm and the OD 280nm of RNA sample, as follows after diluting 50 times with 98 μ l 0.1%DEPC water
Calculate RNA concentration and purity:
=260nm × 50 × 0.04 OD RNA concentration (μ g/ μ l)
RNA purity=OD 260nm/OD 280nm
Ratio is close or larger than 1.8, illustrates that RNA purity is higher, not the remnants of protein；Ratio is less than 1.6, explanation
There is the pollution such as protein, phenol in RNA sample；
B) denaturing formaldehyde gel electroresis appraisal RNA mass: 1% denaturing formaldehyde glue of preparation → take on 5 μ l of RNA sample and RNA
After sample buffer 1:1 is mixed, 65 DEG C of water-baths denaturation 5min → in 1 × MOPS electrophoretic buffer are until electrophoresis to satisfaction (50V),
It is observed under ultraviolet lamp, it is seen that tri- band of 28S, 18S, 5S.
3) real-time quantitative polymerase chain reaction (Real-time PCR).
A) cDNA is synthesized: establishing 20 μ l reverse transcription reaction systems: 5 × Reaction mix, 4 μ l, Maxima enzyme
2 μ l, Template RNA of mix 1 μ l (5 μ g), Nuclease-free water 13 μ l, PCR instrument reverse transcription 30min；
B) primer sequence:
| NONRATT021972 | Upstream: | 5'-GATTAGGAGCAGTACGGTTCA-3'； |
| Downstream: | 5'-TTGTTTGTTTGTTTGGGACA-3'； | |
| Pannexin-1 | Upstream: | 5'-CCCTCTGGTCTGCTCTGTGTC-3'； |
| Downstream: | 5'-GGGGGTCCAGGTCCGTCTCT-3'； |
| IRS1 | Upstream: | 5'-CTCTACACCCGAGACGAACAC-3'； |
| Downstream: | 5'-TGGGCCTTTGCCCGATTATG-3'； | |
| β-actin | Upstream: | 5'-GCTCTTTTCCAGCCTTCCTT-3'； |
| Downstream: | 5'-CTTCTGCATCCTGTCAGCAA-3'； |
C) Real-time PCR is detected, 20 μ l systems: Power10 μ l of Green PCR Master Mix,
Each 1 μ l, cDNA1 μ l, Nuclease-free water of 10 μM of upstream and downstream primers is settled to 20 μ l.It mixes, is placed in ABI
7500PCR instrument is measured using SYBR-green method.Reaction condition is 95 DEG C of 10min, 95 DEG C of 15sec, 60 DEG C of 1min, 40
Circulation.And the specificity of amplification is determined by the solubility curve of analysis product.
12. western blot.
1) prepared by protein sample.
A) the 0.1M PBS for taking each group rat Superior cervical sympathetic ganglia to be put into pre-cooling is washed 3 times；
B) 400 μ l detergents containing PMSF are added in tissue to split in the homogenizer of liquid, grind on ice, pulverizes tissue as far as possible,
1h is cracked on ice；
C) lysate is moved in 1.5ml EP pipe, 4 DEG C, 12000rpm, is centrifuged 10min, supernatant is taken to manage in 0.5ml EP
In, be added 5 × loading buffer, then plus 10%DTT, boiling water boiling 5min, packing, be placed in -20 DEG C of preservations.
2) PAGE gel.
A) 5% or 10% separation gel 10ml:
| Distilled water | 5.7ml (5% separation gel) 4ml (10% separation gel)； |
| 30% acrylamide | 3.3ml (10% separation gel)；1.6ml (5% separation gel) |
| 1.5mol/L Tris(pH 8.8) | 2.5ml |
| 10%SDS | 100μl |
| 10% ammonium persulfate | 100μl |
| TEMED | 4μl |
B) 5% concentration glue of preparation:
| Distilled water | 2.7ml |
| 30% acrylamide | 0.67ml |
| 1.0mol/L Tris(pH 6.8) | 0.5ml |
| 10%SDS | 40μl |
| 10%AP | 40μl |
| TEMED | 4μl |
3) loading and progress SDS-PAGE Protein Separation electrophoresis.
A) be added 5 × Gel Loading buffer, then plus 10%DTT, 95 DEG C are boiled 5min, are made albuminous degeneration, are carried out before electrophoresis
Loading；
B) SDS-PAGE Protein Separation electrophoresis is carried out, glue 90V, 30min, separation gel 120V, 50min, to bromophenol blue is concentrated
It migrates to separation gel bottom, stops electrophoresis.
4) transferring film: current stabilization 300mA transferring film 90min.
5) immunology detection.
A) after transferring film, film 10min is washed with TBST；
B) it closes: with 5% skimmed milk power confining liquid (TBST preparation), room temperature closing, yawing 1h；
C) Pannexin-1, IRS1, p-IRS1ser302 primary antibody (1:1000) in rabbit source or the β-in mouse source is added
Actin primary antibody (1:1000): antibody is diluted to working concentration with confining liquid, total volume is 3~5ml.4 DEG C overnight；
D) TBST washes film 3 times, and 10min/ times, yawing；Secondary antibody or the goat anti-mouse source in goat antirabbit source is added
Secondary antibody: secondary antibody working solution (is diluted with confining liquid with 1:5000), room temperature yawing be incubated for 1h；
E) TBST washes film 4 times, and 10min/ times, yawing；The detection of immune complex: film reacts 5min in ECL, and darkroom exposes
Light, development and fixing.
6) photodensitometry: purpose band is analyzed by Image-Pro Plus image analysis system and is measured in X-ray film
OD value, with the expression quantity of its albumen of the OD value markization of each group β-actin band.
13. statistical method.
Experimental data is for statistical analysis with Excel and 11.5 statistical software of SPSS, and each experimental group data are with mean ± mark
Standard accidentally indicates that otherness uses variance analysis between each group, and comparison among groups use least significant difference (LSD), and p < 0.05 is aobvious
Write sex differernce.
Two, result.
1. the variation of blood glucose.
Each group rat fasting blood-glucose (FBG), postprandial blood sugar (PBG) are without significant difference (p > 0.05), data when experiment starts
Terminal column goes out.Diabetic model group rat the 5th weekend (feeding surrounding through high glucose and high fat) fasting blood-glucose, postprandial blood sugar and normal control
Group is compared and is increased but without significant statistical difference (p > 0.05) (being shown in Table 1).8th weekend (mould and siRNA handle
Diabetic model group rat fasting blood-glucose and postprandial blood sugar are above Normal group (p < 0.01) after a week)；With diabetes mould
Type group is compared with siRNA control group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat
Fasting blood-glucose and postprandial blood sugar decrease (p < 0.05).SiRNA control rats fasting blood-glucose and postprandial blood sugar with
Diabetic model group is higher than Normal group (p<0.01) (being shown in Table 1) compared to no significant difference (p>0.05).
Table 1 tests the variation of the 5th weekend and the 8th weekend each group rat blood sugar
Every class value is mean ± standard deviation；N is every group of rat quantity；**P < 0.01 compared with the control group,#The He of p < 0.05##p
Compared with < 0.01 adds out-of-order siRNA negative control group with diabetic model group and diabetes model.
2. the variation of blood pressure.
Each group rat systolic pressure (SBP) when experiment starts, diastolic pressure (DBP) and mean arterial pressure (MBP) comparing difference without
Unified meter learns meaning (p > 0.05), and data terminal column goes out.Diabetic model group rat the 5th weekend (feeding surrounding through high glucose and high fat) is received
Contracting pressure, diastolic pressure and mean arterial pressure increased compared with Normal group but without significant statistical difference (p > 0.05) (see
Table 2).8th weekend (mould and siRNA processing after a week) diabetic model group rat systolic pressure (SBP), diastolic pressure
(DBP) and mean arterial pressure (MBP) is above Normal group (p < 0.05)；It is compareed with diabetic model group and siRNA
Group is compared, the systolic pressure (SBP) of long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat, diastolic pressure
(DBP) and mean arterial pressure (MBP) reduces (p < 0.05).SiRNA control rats systolic pressure (SBP), diastolic pressure (DBP)
With mean arterial pressure (MBP) compared with diabetic model group without significant difference (p>0.05), but be higher than Normal group (p<
0.05) (3 are shown in Table).
Table 2 tests the variation of the 5th weekend each group rat blood pressure
Every class value is mean ± standard deviation；N is every group of rat quantity.
Table 3 tests the variation of the 8th weekend each group rat blood pressure
Every class value is mean ± standard deviation；N is every group of rat quantity；*P < 0.05 compared with the control group,#P < 0.05 and glycosuria
Disease model group and diabetes model add out-of-order siRNA negative control group to compare.
3. the variation of Electrocardiograph index.
Compared with Normal group, diabetic model group rat the 5th weekend (feeding surrounding through high glucose and high fat) heart rate (HR)
Accelerated, QT interphase (QT interval) is extended, but without significant statistical difference (p > 0.05) (being shown in Table 4).8th week
End (mould and siRNA processing after a week) diabetic model group rat heart rate be higher than Normal group (p < 0.01), QT
Interphase is longer than Normal group (p < 0.01).Compared with diabetic model group and siRNA control group, long non-coding ribose core
The heart rate of sour NONRATT021972 siRNA processing group rat reduces (p < 0.01), QT interval reduction (p < 0.01).It is small dry
RNA control rats heart rate, QT interphase are disturbed compared with diabetic model group without significant difference (p > 0.05), but and Normal group
It compares, increased heart rate, QT interval prolongation (p < 0.01) (being shown in Table 5).
Table 4 tests the variation of the 5th weekend each index of each group Rat Ecg
Values are means±SEM；n is the number of the rats in each group.
Table 5 tests the variation of the 8th weekend each index of each group Rat Ecg
Every class value is mean ± standard deviation；N is every group of rat quantity；*P < 0.05 compared with the control group,#P < 0.05 and glycosuria
Disease model group and diabetes model add out-of-order siRNA negative control group to compare.4. heart rate variability.
HRV frequency-domain analysis the result shows that, compared with Normal group, the 5th weekend of diabetic model group rat is (through high sugar
High fat diet surrounding) general power (TP), very low frequencies power (VLF), low frequency power (LF) and high frequency power (HF) declined, and it is low
The ratio between frequency power and high frequency power (LF/HF) are increased, but without significant statistical difference (p > 0.05) (being shown in Table 6).8th weekend
(mould and siRNA processing after a week) diabetic model group rat TP, VLF, LF and HF lower than Normal group (p <
0.01), LF/HF is higher than Normal group (p < 0.01).Compared with diabetic model group and siRNA control group, long non-volume
TP, VLF, LF and HF of code ribonucleic acid NONRATT021972 siRNA processing group rat increase (p < 0.05), LF/HF drop
Low (p < 0.05).SiRNA control rats TP, VLF, LF HF and LF/HF are compared with diabetic model group without significant difference
(p>0.05), but there are notable difference (p<0.01) (being shown in Table 7) with Normal group.
Table 6 tests the variation of the 5th weekend each group rat heart rate variability
Every class value is mean ± standard deviation；N is every group of rat quantity.
Table 7 tests the variation of the 8th weekend each index of each group rat heart rate variability
Every class value is mean ± standard deviation；N is every group of rat quantity；**P < 0.01 compared with the control group,#The He of p < 0.05##p
Compared with < 0.01 adds out-of-order siRNA negative control group with diabetic model group and diabetes model.5. arterial pressure pressure is quick
Perception.
Compared with Normal group, diabetic model group rat the 5th weekend (feeding surrounding through high glucose and high fat) arterial pressure
Pressure sensibility decreases, but without significant statistical difference (p > 0.05) (being shown in Table 8).8th weekend (mould and small interference
RNA processing after a week) diabetic model group rat artery blood pressure pressure sensibility (BRS) be lower than Normal group (p < 0.05).
Compared with diabetic model group and siRNA control group, at long non-coding ribonucleic acid NONRATT021972 siRNA
The arterial pressure pressure sensibility of reason group rat increases (p < 0.05), but is still below Normal group；SiRNA control group is big
Mouse arterial pressure pressure sensibility (BRS) compared with diabetic model group without significant difference (p > 0.05), but significantly lower than normal
Control group (p < 0.05) (being shown in Table 8).
Table 8 tests the variation of the 5th weekend and the 8th weekend each group rat artery blood pressure pressure sensibility
| 5 weeks | 8 weeks | |
| Control | - 2.43 ± 0.53 (n=5) | - 3.76 ± 0.13 (n=10) |
| Diabetes model | - 2.31 ± 0.32 (n=5) | -1.73±0.21*(n=10) |
| The small interference of diabetes model+NONRATT021972 | - 2.25 ± 0.65 (n=5) | -3.20±0.34#(n=10) |
| Diabetes model+random ordering siRNA negative control | - 2.38 ± 0.53 (n=5) | -1.85±0.22*(n=10) |
Every class value is mean ± standard deviation；N is every group of rat quantity；*P < 0.05 compared with the control group,#P < 0.05 and glycosuria
Disease model group and diabetes model add out-of-order siRNA negative control group to compare.6. rat blood serum interleukin-6 (IL-
6)。
8th weekend Normal group, diabetic model group, diabetes model long non-coding ribonucleic acid
NONRATT021972si group and the concentration of diabetes model NCsi group rat blood serum interleukin-6 is respectively as follows: 198.272 ±
17.858pg/ml (n=10), 765.899 ± 18.904pg/ml (n=10), 260.345 ± 17.112pg/ml (n=10),
720.863 ± 18.835pg/ml (n=10) (Fig. 1).Statistical analysis, the 8th weekend diabetic model group rat blood serum leucocyte
- 6 content of interleukin is apparently higher than Normal group (p < 0.01)；Compared with diabetic model group and siRNA control group, length is non-
Encoding ribose nucleic acid NONRATT021972 siRNA processing group rat blood serum Content of IL-6 be substantially reduced (p <
0.01).SiRNA control rats serum Content of IL-6 compared with diabetic model group without significant difference (p >
0.05), but it is higher than Normal group (p < 0.01).
7. rat blood serum tumor necrosis factor-alpha (TNF-α).
8th weekend Normal group, diabetic model group, diabetes model long non-coding ribonucleic acid
NONRATT021972si group and diabetes model NCsi group rat blood serum tumor necrosis factor-alpha be respectively as follows: 176.655 ±
17.854pg/ml (n=10), 723.370 ± 18.965pg/ml (n=10), 309.833 ± 17.170pg/ml (n=10),
698.576 ± 18.891pg/ml (n=10) (Fig. 2).Statistical analysis the 8th weekend diabetic model group rat blood serum tumour is bad
Necrosis factor-alpha content is apparently higher than Normal group (p < 0.01)；It is long compared with diabetic model group and siRNA control group
Non- encoding ribonucleic acid NONRATT021972 siRNA processing group rat blood serum levels of tnf-alpha is substantially reduced
(p<0.01).SiRNA control rats serum fingerprint content is compared with diabetic model group without significance difference
Different (p>0.05), but it is higher than Normal group (p<0.01).
8. rat blood serum norepinephrine (NE).
8th weekend Normal group, diabetic model group, diabetes model long non-coding ribonucleic acid
The norepinephrine of NONRATT021972si group and diabetes model NCsi group rat blood serum is respectively as follows: 68.379 ±
3.685pg/ml (n=10), 159.422 ± 4.213pg/ml (n=10), 82.134 ± 2.274pg/ml (n=10),
148.721 ± 3.532pg/ml (n=10) (Fig. 3).Statistical analysis the 8th weekend diabetic model group rat serum cleans on first kidney
Parathyrine content is apparently higher than Normal group (p < 0.01)；Compared with diabetic model group and siRNA control group, long non-volume
Code ribonucleic acid NONRATT021972 siRNA processing group rat blood serum Noradrenaline Contents be substantially reduced (p <
0.01).SiRNA control rats Serum Norepinephrine content compared with diabetic model group without significant difference (p >
0.05), but it is higher than Normal group (p < 0.01).
9. rat blood serum oxidative stress index.
5th week and the 8th weekend Normal group, diabetic model group, diabetes model long non-coding ribonucleic acid
NONRATT021972si group and diabetes model NCsi group rat blood serum oxidative stress index MDA, GSH-PX and T-NOS value are shown in
Table 9 and table 10；Statistical analysis the 5th weekend each group rat blood serum MDA, GSH-PX and T-NOS content without significant difference (p >
0.05) (9 are shown in Table)；8th weekend (mould and siRNA after a week) diabetic model group rat blood serum MDA is higher than normal
Control group (p < 0.01), GSH-PX and T-NOS are lower than Normal group (p < 0.05).With diabetic model group and siRNA
Control group is compared, long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat blood serum MDA reduction (p <
0.05), GSH-PX and T-NOS increases (p < 0.01)；SiRNA control rats serum MDA, GSH-PX and T-NOS and sugar
Urinating disease model group, there were significant differences (p<0.05) (being shown in Table 10) compared to no significant difference (p>0.05), but in Normal group.
Table 9 tests the variation of the 5th weekend each group rat blood serum oxidative stress index
Every class value is mean ± standard deviation；N is every group of rat quantity.
Table 10 tests the variation of the 8th weekend each group rat blood serum oxidative stress index
Every class value is mean ± standard deviation；N is every group of rat quantity；*The He of p < 0.05**P < 0.01 compared with the control group,#p
< 0.05 He##Compared with p < 0.01 adds out-of-order siRNA negative control group with diabetic model group and diabetes model.10. immune
The double marks of fluorescence.
10.1. the double marks (Pannexin-1 and NeuN) of Superior cervical sympathetic ganglia fluorescence.
Superior cervical sympathetic ganglia cell Pannexin-1 and the double mark results of neuronal marker (NeuN) antibody mediated immunity fluorescence
Show that Superior cervical sympathetic ganglia cell Pannexin-1 and NeuN antibody have coexpression.Diabetic model group rat
Pannexin-1 expression is apparently higher than Normal group (n=5, p < 0.05).With diabetic model group and siRNA control group
It compares, the Pannexin-1 expression of long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat reduces (n=
5,p<0.05)；SiRNA control rats Pannexin-1 expression compared with diabetic model group without significant difference (n=5,
P>0.05), but it is higher than Normal group (n=5, p<0.05) (Fig. 4).
10.2. the double marks (IRS1 and NeuN) of Superior cervical sympathetic ganglia fluorescence.
Superior cervical sympathetic ganglia cell IRS1 receptor and the double mark results of neuronal marker (NeuN) antibody mediated immunity fluorescence are aobvious
Show that Superior cervical sympathetic ganglia cell IRS1 receptor and NeuN antibody have coexpression.Sympathetic nerve on diabetic model group rat neck
Section IRS1 expression of receptor is substantially less than Normal group (n=5, p < 0.05).With diabetic model group and siRNA control group
It compares, long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat Superior cervical sympathetic ganglia IRS1 expression rises
High (n=5, p < 0.05)；SiRNA control rats Superior cervical sympathetic ganglia IRS1 expression nothing compared with diabetic model group
Significant difference (n=5, p>0.05), but it is lower than Normal group (n=5, p<0.05) (Fig. 5).
11. real-time quantitative polymerase chain reaction (Real-time PCR).
11.1. the expression of Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972.
Real-time PCR detects Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972 expression,
Using Normal group long non-coding ribonucleic acid NONRATT021972 relative expression quantity level as reference, Normal group, glycosuria
Disease model group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group and siRNA control group neck are submitted
Sense neuromere long non-coding ribonucleic acid NONRATT021972 expression is respectively 100.0% (n=5), and 255.3 ± 6.9%
(n=5), 89.3 ± 7.1% (n=5) and 245.6 ± 5.2% (n=5).It is sympathetic on diabetic model group rat neck as the result is shown
Neuromere long non-coding ribonucleic acid NONRATT021972 expression is significantly higher than Normal group (n=5, p < 0.01).With glycosuria
Disease model group is compared with siRNA control group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group neck
Upper sympathetic ganglion long non-coding ribonucleic acid NONRATT021972 expression is decreased obviously (n=5, p < 0.01), and jamming effectiveness reaches
To 65% or so；SiRNA control group Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972 expression with
Diabetic model group is higher than Normal group (n=5, p<0.01) compared to no significant difference (n=5, p>0.05).Show
The experiment of body long non-coding ribonucleic acid NONRATT021972 siRNA can effectively reduce sympathetic nerve on diabetes rat neck
Save the level (Fig. 6) of long non-coding ribonucleic acid NONRATT021972 up-regulated expression.
11.2. the expression of Superior cervical sympathetic ganglia Pannexin-1mRNA.
Real-time PCR detects Superior cervical sympathetic ganglia Pannexin-1mRNA expression, with Normal group
Pannexin-1mRNA relative expression quantity level is reference, Normal group, diabetic model group, long non-coding ribonucleic acid
NONRATT021972 siRNA processing group and siRNA control rats Superior cervical sympathetic ganglia Pannexin-1mRNA
Expression is respectively 100.0% (n=5), and 195.3 ± 7.3% (n=5), 115.8 ± 7.8% (n=5) and 188.9 ±
6.9% (n=5).Diabetic model group rat Superior cervical sympathetic ganglia Pannexin-1mRNA expression as the result is shown is significantly higher than
Normal group (n=5, p < 0.01).Compared with diabetic model group and siRNA control group, long non-coding ribonucleic acid
NONRATT021972 siRNA processing group rat Superior cervical sympathetic ganglia Pannexin-1mRNA expression is decreased obviously (n=
5,p<0.01)；SiRNA control rats Superior cervical sympathetic ganglia Pannexin-1mRNA expression and diabetic model group phase
Than no significant difference (n=5, p>0.05), but it is higher than Normal group (n=5, p<0.01).Show long non-coding ribonucleic acid
Diabetes rat Superior cervical sympathetic ganglia Pannexin-1mRNA up-regulated expression can be reduced after NONRATT021972 siRNA
Level (Fig. 7).
11.3. the expression of Superior cervical sympathetic ganglia IRS1mRNA.
Real-time PCR detects Superior cervical sympathetic ganglia IRS1mRNA expression, with Normal group IRS1mRNA
Relative expression quantity level is reference, and Normal group, diabetic model group, long non-coding ribonucleic acid NONRATT021972 are small
RNA interfering processing group and siRNA control rats Superior cervical sympathetic ganglia IRS1mRNA expression are respectively 100.0%
(n=5), 53.6 ± 8.2% (n=5), 94.3 ± 4.7% (n=5) and 57.9 ± 4.9% (n=5).Diabetes as the result is shown
Model group rats Superior cervical sympathetic ganglia IRS1mRNA expression is substantially less than Normal group (n=5, p < 0.01).With diabetes
Model group is compared with siRNA control group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat
Superior cervical sympathetic ganglia IRS1mRNA expresses significantly raised (n=5, p < 0.01)；Sympathetic mind on siRNA control rats neck
Warp knuckle IRS1mRNA is expressed compared with diabetic model group without significant difference (n=5, p > 0.05), but is lower than Normal group (n
=5, p < 0.01).It can be improved after long non-coding ribonucleic acid NONRATT021972 siRNA sympathetic on diabetes rat neck
Neuromere IRS1mRNA lowers the level (Fig. 8) of expression.
12. western blot.
12.1. the protein expression of Superior cervical sympathetic ganglia Pannexin-1.
Western blot result is through β-actin (43KD) markization, with Normal group Superior cervical sympathetic ganglia Pannexin-1
Albumen (48KD) is expressed as reference, the OD value of Superior cervical sympathetic ganglia Pannexin-1 protein expression Normal group,
Diabetic model group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group and siRNA compare component
Not are as follows: 100.0% (n=5), 237.6 ± 7.9% (n=5), 103.2 ± 5.9% (n=5) and 240.1 ± 8.7% (n=5).
Diabetic model group rat Superior cervical sympathetic ganglia Pannexin-1 protein expression is higher than Normal group (n=5, p < 0.01).
Compared with diabetic model group and siRNA control group, at long non-coding ribonucleic acid NONRATT021972 siRNA
Reason group rat Superior cervical sympathetic ganglia Pannexin-1 protein expression is decreased obviously (n=5, p < 0.01)；SiRNA control group
With diabetic model group rat Superior cervical sympathetic ganglia Pannexin-1 protein expression no significant difference (n=5, p > 0.05), but
Higher than Normal group (n=5, p < 0.01) (Fig. 9).
12.2. the protein expression of Superior cervical sympathetic ganglia IRS1 and p-IRS ser302.
Western blot result is after β-actin (43KD) markization, with Normal group Superior cervical sympathetic ganglia IRS1 albumen
(180KD) is expressed as reference, the OD value of Superior cervical sympathetic ganglia protein expression Normal group, diabetic model group,
Long non-coding ribonucleic acid NONRATT021972 siRNA processing group and siRNA control group are respectively as follows: 100.0% (n
=5), 21.2 ± 2.8% (n=5), 95.3 ± 5.8% (n=5) and 25.9 ± 1.8% (n=5).Diabetic model group rat
Superior cervical sympathetic ganglia IRS1 protein expression is lower than Normal group (n=5, p < 0.01)；With diabetic model group and small interference
RNA control group is compared, long non-coding ribonucleic acid NONRATT021972 siRNA processing group rat Superior cervical sympathetic ganglia
IRS1 protein expression is significantly raised (n=5, p < 0.01)；SiRNA control group rat neck compared with diabetic model group is submitted
Feel neuromere IRS1 protein expression no significant difference (n=5, p>0.05), but lower than Normal group (n=5, p<0.01) (figure
10)。
Western blot result is after IRS1 (180KD) markization, with Normal group Superior cervical sympathetic ganglia p-
IRS1ser302 albumen (180KD) is expressed as reference, the optical density of the p-IRS1ser302 protein expression of Superior cervical sympathetic ganglia
Value is in Normal group, diabetic model group, long non-coding ribonucleic acid NONRATT021972 siRNA processing group and small
RNA interfering control group is respectively as follows: 100.0% (n=5), 308.4 ± 10.8% (n=5), 103.3 ± 8.8% (n=5) and
325.9 ± 8.9% (n=5).The expression of diabetic model group rat Superior cervical sympathetic ganglia p-IRS1ser302 albumen is higher than just
Normal control group (n=5, p < 0.01)；Compared with diabetic model group and siRNA control group, long non-coding ribonucleic acid
The expression of NONRATT021972 siRNA processing group rat Superior cervical sympathetic ganglia p-IRS1ser302 albumen is decreased obviously
(n=5, p < 0.01)；SiRNA control group rat Superior cervical sympathetic ganglia p-IRS1ser302 compared with diabetic model group
Protein expression no significant difference (n=5, p>0.05), but be higher than Normal group (n=5, p<0.01) (Figure 10).
The IRS1 that the research discovery in applicant laboratory passes through high flux gene chip Superior cervical sympathetic ganglia as the result is shown
It is significant after Superior cervical sympathetic ganglia long non-coding ribonucleic acid NONRATT021972 clpp gene drops respectively with Pannexin-1
Upper reconciliation down-regulated gene.Show diabetes Superior cervical sympathetic ganglia long non-coding ribonucleic acid through further function assessment experimental verification
NONRATT021972 high expresses adjustable Superior cervical sympathetic ganglia Pannexin-1 and IRS1 etc. and neuronal cell growth and inflammation
Property related gene expression, participate in diabetic heart autonomic nervous function damage mediate pathological change.Long non-coding ribose core
Sour NONRATT021972 siRNA is by reducing Superior cervical sympathetic ganglia Pannexin-1 and IRS1 expression, to mitigate sugar
The damage for urinating sick heart autonomic function generates preventive and therapeutic effect to the abnormal of diabetic heart autonomic function.
Claims (1)
1. long non-coding ribonucleic acid NONRATT021972 siRNA treats diabetes complicated cardiac autonomic nervous in preparation
Application in the drug of disease, the long non-coding ribonucleic acid NONRATT021972 are described as shown in SEQ ID NO:3
SiRNA as shown in SEQ ID NO:1 and SEQ ID NO:2.
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Citations (1)
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| CN104147345A (en) * | 2014-07-28 | 2014-11-19 | 山东中医药大学附属医院 | Traditional Chinese medicinal composition for treating cardiovascular autonomic neuropathy |
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| CN104147345A (en) * | 2014-07-28 | 2014-11-19 | 山东中医药大学附属医院 | Traditional Chinese medicinal composition for treating cardiovascular autonomic neuropathy |
Non-Patent Citations (4)
| Title |
|---|
| Effects of neferine on CCL5 and CCR5 expression in SCG of type2 diabetic rats;Li G et al;《Brain Research Bulletin》;20130131;第90卷;79-87 * |
| IRS proteins and the common path to diabetes;White MF;《AM J Physiol Endocrinol Metab》;20020930;第283卷(第3期);E413-22 * |
| 糖尿病并发症防治药物研究进展;王雅娟 等;《中国新药杂志》;20051231;第14卷(第6期);678-682 * |
| 长非编码RNA与人类疾病调控机制的研究进展;樊波 等;《中国药理学通报》;20121114;第29卷(第12期);1629-1633 * |
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