CN113893334A

CN113893334A - Sevoflurane influence inhibitor based on cAMP/PKA-CREB-BDNF signal pathway and application thereof

Info

Publication number: CN113893334A
Application number: CN202111217376.4A
Authority: CN
Inventors: 邱颐; 王晓冬; 王莹; 吴育林; 付学强; 王彩霞; 丁玉美; 安敏
Original assignee: Second Affiliated Hospital Of Inner Mongolia Medical University
Current assignee: Second Affiliated Hospital Of Inner Mongolia Medical University
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2022-01-07

Abstract

The invention relates to a sevoflurane influence inhibitor based on a cAMP/PKA-CREB-BDNF signal pathway and application thereof; the inhibitor is a combination of one or more of cAMP, PKA, BDNF, CREB against PND resulting from the use of sevoflurane. The inhibitor is a combination of one or more of BDNFmRNA, CREBmRNA for PND resulting from the use of sevoflurane. The sevoflurane concentration is not less than 3%. The use of the inhibitor as a sevoflurane-induced PND therapeutic.

Description

Sevoflurane influence inhibitor based on cAMP/PKA-CREB-BDNF signal pathway and application thereof

Technical Field

The invention relates to the field of senile dementia and sevoflurane influence regulation, in particular to a sevoflurane influence inhibitor based on a cAMP/PKA-CREB-BDNF signal pathway and application thereof.

Background

The operative neurocognitive dysfunction (PND) is characterized in that patients without preoperative psychogenic dysfunction have reversible mental disorders in many aspects such as memory, mental concentration and orientation force for weeks or months after anesthesia operation, and specifically, the reversible mental disorders are manifested as cognitive dysfunction, impaired memory, anxiety, personality changes, confusion and the like. If the improvement and the intervention are not timely, the cognitive change can be further developed into irreversible cognitive change and even into senile dementia. A number of epidemiological studies have shown that: PND is mainly concentrated in elderly patients over 65 years of age in age distribution, with incidence rates higher than severe post-operative complications such as myocardial infarction and respiratory failure, and in some specific patients (e.g. cardiac surgery) PND incidence rates of up to 50% not only extend patient hospitalization, increase medical costs, and even decrease postoperative self-care and quality of life, complications and mortality in elderly patients, but also increase personal economic burden and social medical investment, accounting for about 20-40 billion dollars in medical costs for PND in the united states in 1999, and about 60 billion dollars in the increase of numbers by 2006. With the increasing annual proportion of the aged patients who need to receive surgical treatment, the appearance of PND of the aged patients after surgery is more common, so that the PND solving problem has great significance for improving public health and social economy. However, at present, the exact etiology and pathogenesis of PND are not clear, and China has not been paid attention until the last decade. Moreover, the current research can not completely and reasonably explain the pathogenesis and the disease progression of the PND, and an effective treatment method is also lacking clinically, so the research on the PND is still a hotspot and difficulty in the field of anesthesiology.

Current research suggests that old age and narcotics are the major risk factors for PND, especially inhalation narcotics are more likely to trigger PND than intravenous narcotics.

Sevoflurane is one of the most commonly used inhalation anesthetics in clinical practice, and its effect on the central nervous system has not been determined. Experiments have shown that sevoflurane can cause cognitive impairment in rats, and recently, experiments have shown that the intoxication concentration of sevoflurane in rats is about 2.4% at the Minimum Alveolar Concentration (MAC) and about 1.3% at 0.65MAC, which is a more common intoxication dose for flaxene]The inhalation of sevoflurane can improve the learning and memory functions of rats, while the mechanism of the influence of sevoflurane on the cognitive function is not clear, and the following main mechanisms are approximately found in the current clinical research and animal experiments: 1) affecting neurotransmitters and receptors, we successfully detected up-regulation of 5-HT1A, 5-HT3 receptor expression in rat hippocampal tissue after sevoflurane anesthesia in previous studies. 2) Inhibition of long-term potentiation (LTP). 3) Promoting nerve cell apoptosis. 4) Other mechanisms are as follows: such as sevoflurane, can increase the level of beta amyloid by altering the production of precursor amyloid, resulting in neurotoxicity or H in peripheral lymphocytes₂0₂Increased production of sevoflurane damages the mitochondrial membrane, mediates the toxic effects of sevoflurane on cells, and the like. Although a large number of receptors, molecules and seven have been found in the studyThe general anesthesia effect of halothane can be related, but the dispersed results are difficult to be organically related, at present, the research considers that the single theory of membrane lipid solubility, ion channel, receptor and gene and the like can not completely explain the complex effect of general anesthesia (especially inhalation narcotic), the general anesthesia medicine has the effect on the central nervous system in multiple directions and multiple target points, the intracellular signal transduction system has rapid change, and can organically connect and integrate a large number of signals, thereby being the main direction for researching the action mechanism of the future medicine. The influence of the inhalation anesthesia process on the activation of signal transduction molecules in the brain is observed, and the general anesthesia mechanism is explored from a new view angle.

Cyclic adenosine monophosphate response element binding protein (CREB) is an important nuclear transcription factor regulating the central nervous system, and it regulates transcription by autophosphorylation. In several studies, a significant decrease in hippocampal CREB levels was demonstrated in rats after sevoflurane inhalation, and it is believed that long-term inhibition of CREB activation may be one of the molecular mechanisms of cognitive and learning memory impairment after general anesthesia, but it is not known how sevoflurane acts on CREB to cause cognitive changes. CREB is regulated by various signal transduction pathways, wherein cyclic AMP (cAMP)/protein kinase A (protein kinase A, PKA) -CREB signal pathways are activated by the action of neurotransmitters and receptors, and are widely applied to the research of long-term memory, and in addition, the research on learning memory of mammals and vascular endothelial growth factor and the like discover the participation of the signal system; ca²⁺A calmodulin dependent protein kinase (CaMK) -CREB signaling pathway, which is mainly used for the research of inhibiting the formation of long-term memory and is also found to participate in the regulation research of the development and the function of the nervous system by the brain neurotrophic factor in recent years; PI (proportional integral)₃the/Akt signaling pathway has been shown to be involved in regulating many cellular processes, including survival, apoptosis, proliferation and metabolism;

brain Derived Neurotrophic Factor (BDNF), a protein synthesized in the brain, widely distributed in the central nervous system, activated by phosphorylated CREB, has the function of promoting the survival and differentiation of central neurons, and plays an important role in improving cognitive dysfunction; in addition, BDNF can regulate synaptic transmission and synaptic plasticity, promote and maintain the LTP of synaptic transmission before and after the synapse, and play a vital role in the formation and maintenance of learning and memory functions; studies have shown that a decrease in its level can cause neuronal degeneration. In animal experiments, pharmacological experiments (reducing the level of BDNF such as antisense oligonucleotides and BDNF antibodies through the administration of drugs) and gene knockout BDNF can lead to the development of learning and memory dysfunction of animals, and the physiological functions of BDNF can exactly summarize the mechanism of the influence of the current sevoflurane on the cognitive function, however, no research for the change of the BDNF caused by the current sevoflurane is proved.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a sevoflurane influence inhibitor based on a cAMP/PKA-CREB-BDNF signal pathway and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

sevoflurane-affected inhibitors based on the cAMP/PKA-CREB-BDNF signaling pathway; the inhibitor is a combination of one or more of cAMP, PKA, BDNF, CREB against PND resulting from the use of sevoflurane.

Further, the inhibitor is a combination of one or more of BDNFmRNA, CREBmRNA for PND resulting from the use of sevoflurane.

Further, the sevoflurane concentration is not less than 3%.

In the inhibitor effect verification process, the adopted qPCR detection primers are as follows:

in the effect verification process of the inhibitor, the PCR reaction is specifically set as follows:

pre-denatured Cycle 1 (1X)

Step 1:95.0℃ for 05:00.

PCR Cycle 2 (40X)

Step 1:95.0℃ for 00:10.

Step 2:60℃ for 00:30.

Data collection and real-time analysis enabled.

Dissolution curve Cycle 3 (71X)

Step 1:60.0℃-95.0℃ for 00:30.

Increase set point temperature after cycle 2by 0.5℃

Melt curve data collection and analysis enabled.

Further, the combination of one or more of cAMP, PKA, BDNF and CREB is used as a sevoflurane induced PND treatment drug.

Further, the combination of one or more of BDNFmRNA and CREBmRNA is used as a sevoflurane induced PND treatment drug.

Further, the sevoflurane concentration is not less than 3%.

Compared with the prior art, the invention has the beneficial effects that:

the invention relates to a sevoflurane influence inhibitor based on a cAMP/PKA-CREB-BDNF signal pathway and application thereof; verifying whether different concentrations of sevoflurane have concentration-related dual effects on the cognitive function of the aged rats; on the basis of an animal model, a western-blot method is adopted to detect the expression changes of cAMP, PKA, CREB and BDNF in a hippocampal tissue, RT-PCR is adopted to detect the expression changes of CREB and BDNFmRNA, the relevant mechanism of the influence of sevoflurane on the cognitive function is discussed, the guiding significance is provided for the clinical use dosage of sevoflurane, and the research foundation is laid for further discussing the mechanism of the influence of sevoflurane on the cognitive function. Has important clinical significance for clarifying the action mechanism in the occurrence process of PND caused by sevoflurane and preventing and treating PND; the PND of the organism caused by the use of sevoflurane can be effectively inhibited by up-regulating cAMP, PKA, BDNF and CREB expression in tissues and BDNFmRNA and CREBmRNA expression. Improve the postoperative recovery condition of the old. Reduces the risk of PND and has good clinical application prospect.

Drawings

FIG. 1 is a gray scale representation of BDNF of different groups;

FIG. 2 is a gray scale representation of BDNF of different groups; note: p <0.05 compared to group C;

FIG. 3 is a gray scale representation of different groups of CREB;

FIG. 4 is a graph of expressed gray scale analysis of different groups of CREB;

note: p <0.05 compared to group C;

FIG. 5 is a gray scale representation of different groups of PKA;

FIG. 6 is a technical roadmap for the present invention.

FIG. 7 is a histogram of the expression analysis of the genes of interest CREB, BDNF, TrkB and CaMKIV.

Detailed Description

The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:

test example: sevoflurane influence inhibitor based on cAMP/PKA-CREB-BDNF signaling pathway and uses thereof, as shown in FIGS. 1-7

(1) Establishing a rat model of PND (PND) after sevoflurane anesthesia, and observing the influence of sevoflurane with different concentrations on the cognitive function of aged rats

Selecting aged (18-20 months old) wister rats as research objects, carrying out anesthesia treatment on sevoflurane with different concentrations, testing the ethological ability of the rats by using a morris water maze, and carrying out pathological observation on hippocampal tissues of the rats so as to determine whether the influence of the sevoflurane with different concentrations on the cognitive function has concentration-related dual effects.

(2) On the basis of animal models, the influence of sevoflurane on cAMP/PKA-CREB-BDNF signal pathways is researched.

The expression changes of cAMP, PKA, CREB and BDNF proteins in hippocampal tissues are detected by a westen-blot method, the expressions of CREB and BDNFmRNA are detected by PCR, and the molecular mechanism of the influence of sevoflurane on the cognitive function is discussed.

The research finds that

(1) Verifying whether different concentrations of sevoflurane have concentration-related dual effects on the cognitive function of the aged rats;

(2) the method has the advantages that the relevant mechanism of the influence of the sevoflurane on the cognitive function is discussed, the guiding significance is provided for the clinical use dosage of the sevoflurane, and the research foundation is laid for further discussing the mechanism of the influence of the sevoflurane on the cognitive function.

(3) The influence of different concentrations of sevoflurane on postoperative cognitive function is clarified.

(4) The expression change of a cAMP/PKA-CREB-BDNF signal pathway in the occurrence and development process of sevoflurane-induced PND is determined, and the relevant mechanism of the influence of sevoflurane on the cognitive function is discussed

And establishing an animal model of the PND after the sevoflurane anesthesia, and identifying whether the model is successful.

(1) Grouping experiments: the wister rats were randomly divided into 5 groups (n-15) according to the random number table method, each of which was an empty self-control group (group C), a 1% sevoflurane group ((group S1), a 2% sevoflurane group (group S2), a 3% sevoflurane group (group S3), and a 4% sevoflurane group (group S4).

(2) Method of producing a composite material

1) Establishment of rat sevoflurane inhalation model

During the experiment, the rats are placed in a self-made transparent anesthesia box (the size of 50cm multiplied by 40cm is made of transparent glass, soda lime is paved at the bottom of the box), one side hole of the anesthesia box is connected with a Drager Fabius anesthesia machine (Drager company, Germany), and one side hole of the anesthesia box is connected with a solar8000M multifunctional monitor (GE company, USA) to monitor the concentration of anesthetic gas. Respectively sucking mixed gas of sevoflurane and oxygen (2L/min) with different concentrations (1%, 2%, 3%, 4%) for 6h, and sucking 2L/min oxygen for 6h in the group C under the same environment, then placing the group C back to a constant-temperature animal room, freely taking food, taking 8 hippocampus of each group 24h after sucking, cutting off the heads of the groups on ice, and taking the remaining part for water maze test.

2) Morris Water maze test

The Morris water maze is a circular iron sheet pool with a diameter of 120Cm and a water depth of 50 Cm. The pool wall is provided with 4 water inlet points of east, south, unitary and north, the connecting lines of south and north and east unitary divide the pool into 4 quadrants which are respectively called as quadrant 1 (SE), quadrant 2 (NE), quadrant 3 (N W) and quadrant 4 (SW), a platform which has the same color with the background of the pool is placed at the position which is separated from the pool wall 24Cm in the middle of quadrant 4, and the platform is lower than the water surface 3 Cm. Placing a coloring agent into water, keeping a reference object outside the water maze unchanged, and controlling the water temperature to be about (24+1) DEG C. Rats were placed in a pool without a platform for free swimming 1d prior to training to acclimate and to reject floating and resting rats. (1) Positioning navigation experiment test: the rats are placed into water facing to the pool wall, the time from water entering to hidden platform finding is recorded as a latent period, if the platform can not be found, the rats are guided to stay on the platform for several seconds, the latent period is recorded as 60s, and the rats are respectively placed into water from 4 quadrants every day for training and recording for 5 d. The average incubation period of the day is taken as the achievement in daily training, the swimming track is taken as the total course, and the average value is taken as the achievement. (2) And (3) a space exploration experiment, namely removing the platform in the swimming training on the 6 th day, and recording the times of the rat passing through the position of the platform within 60s and the activity time of the quadrant of the platform.

Pathological observation and detection of main elements on cAMP/PKA-CREB-BDNF signal transduction paths are carried out on rat hippocampal tissues.

(1) Tissue sampling and treatment:

after anesthesia by injecting 10% chloral hydrate 0.3ml/100g into abdominal cavity, taking brain immediately, separating bilateral hippocampus on ice, weighing the standard wood, and storing in liquid nitrogen in a freezing tube for later use.

(2) Pathological observation of hippocampal tissues:

the hippocampal tissues were sectioned with conventional paraffin at a slice thickness of 5 μm, were subjected to Niger staining according to a conventional method, and were subjected to pathological observation using an electron microscope.

(3) Detection of cAMP, PKA, CREB, BDNF levels using immunoblotting (western blot): taking hippocampal tissue, adding lysis solution (1mg:4ul) at low temperature for tissue lysis, homogenizing, centrifuging, and extracting supernatant to obtain total protein extract, and performing protein quantification by BCA method. The whole protein extract was electrophoresed, transferred to a membrane, blocked, and incubated overnight at 4 ℃ in the corresponding primary-resistant diluent with slow shaking. The membrane was washed, placed in the corresponding secondary antibody dilution and incubated for 1h at room temperature. Washing the membrane, incubating the substrate, and performing chemiluminescence coloration.

(4) RT-PCR detection of CREBmRNA and BDNFmRNA expression: the first step is as follows: total RNA extraction Total RNA was extracted by the isothiocyanate method, and the tissue was homogenized in lml Trizol reagent, centrifuged, mixed with isopropyl alcohol, and incubated at room temperature for 10 min. Centrifuging at 4 deg.C for 15min, discarding supernatant, washing precipitate with 70% ethanol, and dissolving with 20ul DEPC water. After purification, the RNA pellet was dissolved in DEPC-treated water, the RNA concentration was measured with a spectrophotometer, and the RNA pellet was stored at-80 ℃ for further use.

The second step is that: reverse transcription to synthesize cDNA: to 50ul of reaction volume was added 5ug total RNA, 10ul of 5 Xreaction buffer, 5ul of 10mmoL/L dNTPs, 0.5ul of RNase (Promega,40U/mL), oligo (dT)_12-18(Invitrogen)0.25ug, reverse transcriptase (M-MLV, Invitrogen,200U/ml)2ml, 0.1mol/L dithiothreitol 0.5ml, incubated at 37 ℃ for 1h, then heated at 65 ℃ for 5min, stored at-20 ℃.

The third step: polymerase chain reaction amplification: to a reaction volume of 25ul were added 0.1ug of synthesized cDNA, 2.5ul of 10 XPCR buffer, 2.5ul of dNTPs (2mmol/L), 2.5ul of magnesium chloride (25mmol/L), 1 ul of each primer (20pmol/ml), and 0.5ul of Taq DNA polymerase (Takara, 5U/. mu.l). Thermal cycling was performed using a PTC-100 polymerase chain reaction instrument (MJResearch).

The primer sequence is as follows: BDNF (F)5-GCT GCT GGA TGA GGA CCA GA-3

(R)5-CCA AAG GCA CTT GAC TGC TG-3

CREB(F)5-CCA CTC AGC CGG GTA CTA CC-3

(R)5-CAC CAG AGG CAG CTT GAA CA-3

Main experimental results

(1) Animal behavioral testing results:

the escape latency of rats in different groups in the Morris water maze experiment is counted, the result shows that on the 5 th training day, the escape latency time difference of rats in each group has no statistical significance (P >0.05), after modeling, compared with the group C, the groups S1 and S2 have no statistical difference (P >0.05) in the 5 th training latency, and the groups S3 and S4 have prolonged (P <0.05) in the 5 th training latency, and the specific statistical result is shown in Table 1.

TABLE 1 comparison of escape latencies of two groups of aged rats (` x. + -. s)

Note: p <0.05 compared to group C

The number of primary platform quadrant crossing times of rats of different groups in the Morris water maze experiment is counted, the result shows that on the 5 th training day, the difference of the primary platform quadrant crossing times of the rats of each group has no statistical significance (P is greater than 0.05), after modeling, compared with the group C, the number of primary platform quadrant crossing times of the rats of S1 and S2 has no statistical difference (P is greater than 0.05) compared with the primary platform quadrant crossing times of the training 5d, the primary platform quadrant crossing times of the rats of S3 and S4 are all reduced (P is less than 0.05), and specific statistical results are shown in a table 2.

TABLE 2 comparison of quadrant crossing times of the IV platform for various groups of aged rats (` x. + -. s)

Note: p <0.05 compared to group C

Statistics is carried out on the residence time of the IV platform quadrant of rats in different groups in the Morris water maze experiment, the results show that on the 5 th day of training, the residence time difference of the original platform quadrant of the rats in each group has no statistical significance (P >0.05), after modeling, compared with the group C, the differences of the groups S1 and S2 have no statistical difference (P >0.05), the residence time of the original platform quadrant of the rats in each group S3 and S4 is shortened (P <0.05) compared with that in the 5 th day of training, and specific statistical results are shown in tables 1-3.

TABLE 3 comparison of quadrant residence time (` x. + -. s) for the IV platform for groups of older rats

Note: p <0.05 compared to group C

(2) western-blot detection result

Results of changes in the expression of BDNF, CREB and p-CREB in rat hippocampal region

And detecting the change of the expression content of the hippocampal tissue protein of each group of rats by using a Western Blot technology. The detection result shows that compared with the control group C, the BDNF protein expression levels of S1 and S2 are not statistically different (P >0.05), the BNDF protein expression levels of S3 and S4 are reduced, and the BDNF expression is reduced along with the increase of sevoflurane concentration, and the difference is statistically significant (P < 0.05). The statistical results are shown in fig. 1 and fig. 2.

Results of changes in rat hippocampal CREB and p-CREB expression

The expression of CREB and phosphorylated CREB (P-CREB) in rat hippocampal tissues is detected, and the result shows that compared with a control group C, the expression levels of CREB and P-CREB proteins of S1 and S2 are not statistically different (P >0.05), the expression levels of CREB and P-CREB proteins of S3 and S4 are reduced, and the expression of CREB phosphorylation is reduced along with the increase of sevoflurane concentration, and the difference has statistical significance (P < 0.05). The statistical results are shown in fig. 3 and 4.

Results of PKA expression changes in rat hippocampal region

By adopting Western Blot to detect PKA in rat hippocampal tissues, compared with the group C, PKA expression in hippocampal neurons of the groups S1 and S2 is gradually reduced after anesthesia, the difference has no statistical significance (P >0.05), PKA expression in the groups S3 and S4 is gradually reduced, and the difference has statistical significance (P <0.05), which is shown in figure 5.

Results of changes in cAMP expression in rat hippocampal region

The Elisa method is adopted to detect the cAMP of the hippocampal tissue, and the result shows that compared with the group C, the cAMP expression changes of the groups S1 and S2 have no statistical difference, the cAMP expression of the groups S3 and S4 is obviously reduced (P <0.05), and the difference has statistical significance, which is shown in Table 4.

Table 4 comparison of cAMP expression in hippocampal tissues of five groups of rats (n ═ 15, x ± s)

Note: in comparison with the group C,^*P<0.05

the results demonstrate that inhalation of high concentrations of sevoflurane can lead to the onset of PND, and the effect on post-operative cognition is exacerbated as the inhaled concentration increases, providing dose guidance for the clinical use of sevoflurane; in addition, sevoflurane was observed in studies to cause down-regulation of the expression of various response elements in the cAMP/PKA-CREB-BDNF pathway in hippocampal neurons, and thus the cAMP/PKA-CREB-BDNF signaling pathway was thought to be involved in the development of PND.

Test example 2:

first, purpose of experiment

Extracting RNA of 15 cases of hippocampal tissues, and detecting background expression of CREB, BDNF, TrkB and CaMKIV genes by qPCR after reverse transcription into cDNA.

Second, Experimental materials

Experimental equipment

TABLE 5 Main Equipment involved in the experiment

Main experimental reagent

TABLE 6 major reagents involved in the experiment

Detecting a sample: 15 examples of Hippocampus tissues

K1 left	Left side of K2	Left side of K3
			1-2	1-4	1-6
2-2	2-4	2-6
			3-2	3-4	3-6
4-2	4-4	4-6

Third, the experimental procedure

1. Extraction of Total RNA from tissues

1) Adding 1ml Trizol into appropriate amount of tissue sample, homogenizing with homogenizer, and standing at room temperature for 5min to completely separate nucleic acid protein complex.

2) Mu.l of chloroform was added to each tube, the eptube was turned upside down by hand for 15s, allowed to stand at room temperature for 3min, at 4 ℃ and 12000rpm, and centrifuged for 15 min.

3) The supernatant was aspirated and transferred to a new 1.5ml eptube, and equal volume of pre-cooled isopropanol at-20 ℃ was added, mixed well and precipitated at-20 ℃ for 30 min.

4) Centrifuging at 4 deg.C and 12000rpm for 10min, removing supernatant, adding 1ml of 4 deg.C pre-cooled 75% ethanol, washing precipitate, centrifuging at 4 deg.C and 10000rpm for 5min, removing supernatant, and drying at room temperature.

5) Mu.l of RNase-free water was added to dissolve completely, and the concentration of extracted RNA was determined by UV analysis.

Reverse transcription of RNA into cDNA

Reverse transcription was performed according to the protocol as follows:

add to RNase-Free PCR tube (20 ul total)

RNA	1μg
		DEPC·H 2O	Make up to 11.0ul

Mixing, incubating at 65 deg.C for 5min, immediately cooling in ice bath at-20 deg.C for 3min, and adding

5×reaction buffer	4.1μl
		RevertAid Reverse Transcriptase(100U/μl)	0.6μl
dNTP mix(10mM)	2.1μl
		Ribolock RNaes Inhibtor(40U/μl)	1.1μl
Oligo(dT)primer(0.2μg/μl)	1.1μl

Incubating at 42 deg.C for 60min, and at 72 deg.C for 10 min; the cDNA was frozen at-20 ℃ or immediately subjected to PCR.

qPCR primer design

Designing and synthesizing qPCR detection primers according to the sequence information of the target gene, wherein the sequences are shown in a table 7;

TABLE 7RT-PCR detection primer information

RT-PCR preliminary experiments

And (3) amplifying a target gene segment to be detected and a housekeeping gene by using a target gene specific primer and an internal reference primer by using the reverse transcribed cDNA as a template, searching for the optimal primer annealing temperature and template amount suitable for a qPCR machine, and readjusting the annealing temperature in the PCR process if a miscellaneous band exists until the miscellaneous band disappears.

qPCR official experiment

1) Under the premise of finding out the optimal primer annealing temperature and template amount in an RT-PCR pre-experiment, 2 xSYBR Green Mix is used for preparing the PCR Mix, and the PCR Mix is calculated and prepared according to the number and the repetition number of samples needing to be loaded on a computer, wherein the system is as follows:

composition (I)	Volume of
		2*SYBR Green Mix	10ul
Primer Mix (2mM)	1μl
		Template (cDNA diluted 25 times)	5μl
Ultrapure water	4μl
		Total volume	20ul

2) Subpackaging into AXYGEN PCR8 connecting tubes, and instantly centrifuging by a miniature centrifuge to mix the PCR system uniformly.

3) Putting the sample into an IQ5 fluorescent quantitative PCR instrument, carrying out SYBR Green fluorescent quantitative PCR to analyze the expression of each gene, and setting the PCR program as follows:

the PCR reaction can be carried out in 2 steps: (the annealing temperature is set by itself according to the Tm value of the primer and the result of the RT-PCR preliminary experiment, and the melting curve can be set to 60-95 ℃.)

Pre-denatured Cycle 1 (1X)

Step 1:95.0℃ for 05:00.

PCR Cycle 2 (40X)

Step 1:95.0℃ for 00:10.

Step 2:60℃ for 00:30.

Data collection and real-time analysis enabled.

Dissolution curve Cycle 3 (71X)

Step 1:60.0℃-95.0℃ for 00:30.

Increase set point temperature after cycle 2by 0.5℃

Melt curve data collection and analysis enabled.

6. And (3) data analysis: relative quantification of gene expression was performed by the Δ Ct method.

Fourth, experimental results

qPCR data analysis

A. Amplification curves of target gene and reference gene

B. Melting curves of target gene and reference gene

Quantitative results analysis of C.qPCR

Data analysis is shown in FIG. 7

The invention (1) establishes a rat model of PND (pnD) after sevoflurane anesthesia, and observes the influence of sevoflurane with different concentrations on the cognitive function of aged rats

Selecting aged (18-20 months old) wister rats as research objects, carrying out anesthesia treatment on sevoflurane with different concentrations (1%, 2%, 3% and 4%), testing the ethological ability of the rats by adopting a morris water maze, and recording the escape latency, the swimming distance, the crossing times of an original platform and the residence time of a second platform quadrant of the aged rats so as to determine whether the influence of the sevoflurane with different concentrations on the cognitive function has concentration-related dual effects.

After modeling is finished, 5 rats are randomly selected from 1d, 3d and 7d for each group to be killed, hippocampal tissues are taken, expression changes of cAMP, PKA, CREB and BDNF proteins in the hippocampal tissues are detected by a western-blot method, the expressions of CREB and BDNFmRNA are detected by RT-PCR, and a molecular mechanism of influence of sevoflurane on cognitive function is discussed.

The sevoflurane influence inhibitor based on a cAMP/PKA-CREB-BDNF signal pathway and application thereof detect the protein and mRNA expression changes of cAMP, PKA, CREB and BDNF in rat hippocampal tissues through RT-PCR and western-blot on the basis of an animal model, and clarify the action and mechanism of PND generation caused by sevoflurane from a cell signal transduction pathway. The research results are as follows: the water maze test shows that compared with the group C, the escape latency of the rats of the group S1 and the group S2 has no statistical significance on the difference of the times of crossing the platform quadrant, the escape latency of the rats of the group S3 and the group S4 is prolonged, and the times of crossing the platform quadrant are reduced (P is less than 0.05); the results of the western-blot assay compared with those of group C, the cAMP, PKA, BDNF and CREB expression in hippocampal tissues of rats of group S3 and group S4 were down-regulated (P <0.05), and there was no statistical difference between groups S1 and S2 and group C. Compared with the group C, the expression of BDNFmRNA and CREBmRNA in hippocampal tissues of rats in the group S3 and the group S4 is reduced (P <0.05) in the RT-PCR detection result, and no statistical difference exists between the group S1 and the group S2 and the group C. The results indicate that high concentrations (greater than 3%) of sevoflurane inhaled lead to the development of PND in elderly rats, the mechanism of which is related to changes in elements of the cAMP/PKA-CREB-BDNF pathway. The PND of the organism caused by the use of sevoflurane can be effectively inhibited by up-regulating cAMP, PKA, BDNF and CREB expression in tissues and BDNFmRNA and CREBmRNA expression. Improve the postoperative recovery condition of the old. Reduces the risk of PND and has good clinical application prospect.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.

Claims

1. Sevoflurane-affected inhibitors based on the cAMP/PKA-CREB-BDNF signaling pathway; characterized in that the inhibitor is a combination of one or more of cAMP, PKA, BDNF, CREB against PND resulting from the use of sevoflurane.

2. The inhibitor according to claim 1, wherein the inhibitor is a combination of one or more of BDNFmRNA, CREBmRNA for PND resulting from the use of sevoflurane.

3. The inhibitor according to claim 1 or 2, characterized in that the sevoflurane concentration is not less than 3%.

4. The inhibitor according to claim 1, wherein in the inhibitor effect verification process, qPCR detection primers are adopted as follows:

5. the inhibitor according to claim 4, wherein in the inhibitor effect verification process, the PCR reaction setup specifically comprises:

pre-denatured Cycle 1 (1X)

Step1:95.0℃ for 05:00.

PCR Cycle 2 (40X)

Step 1:95.0℃ for 00:10.

Step 2:60℃ for 00:30.

Data collection and real-time analysis enabled.

Dissolution curve Cycle 3 (71X)

Step 1:60.0℃-95.0℃ for 00:30.

Increase set point temperature after cycle 2 by 0.5℃

Melt curve data collection and analysis enabled。

6. The use of the inhibitor of claim 1 as a sevoflurane-induced PND treatment.

7. The use of the inhibitor of claim 2 as a sevoflurane-induced PND treatment.

8. The use according to claim 6 or 7, wherein the sevoflurane concentration is not less than 3%.