CN111407782B - Application of strain in preparation of medicine for preventing and/or treating postoperative nerve functional disorder of senile non-cardiac surgery patient - Google Patents

Abstract

The present disclosure discloses the use of a strain for the preparation of a medicament for the prevention and/or treatment of post-operative neurological disorders in elderly non-cardiac surgery patients, wherein the strain is selected from bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis.

Description

Application of strain in preparation of medicine for preventing and/or treating postoperative nerve functional disorder of senile non-cardiac surgery patient

Technical Field

The disclosure belongs to the field of biomedicine, and particularly relates to application of a strain in preparation of a medicine for preventing and/or treating postoperative nerve functional disorder of an elderly non-cardiac surgery patient.

Background

Post-operative neurocognitive dysfunction, including delirium and cognitive dysfunction, is more common in elderly non-cardiac surgery patients. With the unprecedented aging rate of the global population, post-operative neurocognitive function impairment is becoming more prevalent. Postoperative complications arise that increase medical costs, reduce independence from life, and even positively correlate with mortality. Therefore, prevention and treatment of post-operative neurocognitive dysfunction has become one of the targets for improving the quality of medical treatment in the elderly. At present, the prevention and treatment of the postoperative neurocognitive dysfunction mainly depends on the modes of adjusting risk factors, reducing physiological stress, increasing individual reserve capacity, treating symptoms and the like.

However, the high incidence of post-operative neurocognitive impairment in elderly patients remains a significant problem, and there is an urgent need for novel, simple, effective methods of prevention and treatment. In the journal of 2019, A randomised, Double-Blind, Placebo-Controlled Trial of Probiotics to Reduce the sensitivity of the therapy for Patients With Nasophageal Carcinoma, the present inventors disclosed new techniques for preventing and/or treating Oral Mucositis caused by radiotherapy and chemotherapy, and no prior art for preventing and/or treating postoperative neurological disorders in elderly non-cardiac surgery Patients With any bacterial-like drug is currently seen.

Disclosure of Invention

In view of this, the present disclosure discloses the use of a strain selected from bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis for the preparation of a medicament for the prevention and/or treatment of post-operative neurological disorders in elderly non-cardiac surgery patients.

Preferably, the number of viable bacteria of Bifidobacterium longum, Lactobacillus acidophilus and enterococcus faecalis is not less than 1.0 × 10⁷CFU。

Drawings

Fig. 1 to 8 are schematic views of embodiments disclosed in the present disclosure.

Detailed Description

In order to make those skilled in the art understand the technical solutions disclosed in the present disclosure, the technical solutions of the various embodiments will be described below with reference to the embodiments and the related drawings, and the described embodiments are a part of the embodiments of the present disclosure, but not all of the embodiments. Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the disclosure. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It will be appreciated by those skilled in the art that the embodiments described herein may be combined with other embodiments.

In one embodiment, the present disclosure discloses the use of a strain for the preparation of a medicament for the prevention and/or treatment of post-operative neurological dysfunction in an elderly non-cardiac surgery patient, wherein the strain is selected from bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis.

With respect to the above examples, the inventors designed a randomized clinical trial to test whether probiotics comprising the above strains could prevent and/or treat post-operative neurological disorders in elderly non-cardiac surgery patients.

Based on the previous study, we selected as the subject an elderly patient who underwent limited hip or knee replacement surgery or colorectal cancer surgery.

The inclusion criteria were: patients who received limited hip or knee replacement surgery or colorectal cancer (CRC) surgery, age 60-90 years, without immune system disease. Except for patients who meet the following criteria: hospitalization time was less than 7 days; once had suffered from colorectal cancer; perioperative cerebral infarction; requiring emergency surgery (rather than elective surgery) or other types of surgery; multiple surgeries are required during hospitalization; communication impairment before surgery (due to coma, severe dementia, parkinson's disease, severe hearing or vision impairment, language impairment); antibiotics, probiotics or gastrointestinal motility drugs are used 10 days before admission; symptoms of ileus or confirmation of ileus; complete parenteral nutrition is required post-operatively.

Randomizing and masking

Random numbers were generated at a 1:1 ratio by researchers not involved in data management or statistical analysis using SPSS18.0 software. During the study, enrolled patients received probiotic or placebo capsules from nurses according to randomly assigned numbers. During the study period, the investigators responsible for follow-up, surgeons, patients were all concealed from treatment assignment. If the patient's condition suddenly worsens, the attending physician may request that a treatment regimen be published, or that the patient be removed from the study if necessary.

Medicine

Eligible patients were randomized into control and probiotic groups. The probiotic group took probiotic capsules (0.84g) twice daily from admission to discharge. Probiotic capsule (Bifico, Chinese name: Pefilkang, SHANGHAI SINE PHARMACEUTICAL CO. LTD, supra)Sea-letter pharmaceutical factory limited, SFDA approval No.: s10950032) contains Bifidobacterium longum (not less than 1.0 × 10⁷cfu/g), Lactobacillus acidophilus (Lactobacillus lactis) (≧ 1.0 × 10⁷cfu/g), and Enterococcus faecalis (Enterococcus faecium) (. gtoreq.1.0X 10)⁷cfu/g). The control group took placebo capsules according to the same dosing schedule. The placebo capsules contained only starch, but were otherwise similar in shape, size and odor to the Bifico probiotic capsules.

The primary endpoint was the incidence of cognitive dysfunction on day7 post-surgery. Cognitive status was evaluated using a simple Mental State Examination table (MMSE) at admission and on day7 after surgery. Previous studies have shown that the change in MMSE score in alzheimer's patients is typically less than 3 points per year. A significant decline in cognitive performance is generally defined as a change in MMSE score of 3 points or more. Post-operative cognitive dysfunction was defined as a 3 or more point reduction in MMSE score from admission to post-operative day7, according to previous criteria.

Secondary endpoints included changes in plasma interleukin-6 (IL-6) and cortisol, intensity of post-operative pain, post-operative sleep quality, recovery of gastrointestinal function and perioperative fecal flora composition. The pain digital Rating Scale (NRS) was performed once a day7 days after surgery. Patients are asked to score their pain level between 0 and 10 points, with 0 points indicating no pain and 10 points indicating the most severe pain that can be imagined. NRS is considered an effective and reliable pain assessment tool. The sleep quality of the patients was evaluated 7 days after the operation, 0-10NRS once a day, 0 means the worst sleep quality, and 10 means the best sleep quality. Post-operative gastrointestinal function recovery assessment included days or hours to first exsufflation (d/h), days or hours to first fluid diet (d/h), days or hours to first solid diet (d/h), days to first bowel movement (d).

The research assistant is responsible for evaluating the patient's status from admission to discharge, including cognitive testing. The investigator was trained to manage all tests and was supervised by an experienced investigator.

Other endpoints include post-operative hospital stays, cumulative time of antibiotic treatment and hospital stays.

Blood samples were collected in the morning, on day1 and on days 5-7 of the surgery, respectively, and standard laboratory tests for plasma IL-6 and cortisol were performed. Briefly, blood samples were centrifuged at 1000g for 10min at 4 ℃ and plasma was stored at-80 ℃ until analysis. Two-step sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect IL-6 and cortisol levels in plasma.

Fecal samples were taken 1 day before treatment and 5-7 days post-surgery for bacterial 16S ribosomal RNA (rRNA) gene sequencing. Participants were asked to sample their stools with a cotton swab according to detailed printed instructions. Briefly, stool samples were placed in ice boxes (mostly within 4 hours after collection) and frozen at-80 ℃ until analysis. Microbial DNA was extracted from each sample using the QIAamp DNA pool Mini kit (Qiagen, Hilden, Germany). The V3-V4 hypervariable region of 16S rRNA was amplified from genomic DNA using primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC). Amplicons were quantified using a qubit quantification system (Thermo Scientific, Wilmington, DE, US) and amplicon sequencing using the Illumina MiSeq system (Illumina inc., San Diego, CA, USA). Fastq files are demultiplexed using MiSeq controller software (Illumina). Sequences were tailored for amplification primers, diversity intervals and sequencing adapters, pooled pairings, and then quality screened with USEARCH. UPARSE is used for clustering of open reference operation classification units (OTUs) and is greater than or equal to 97%. The OTU was analyzed using phylogeny and OTU application in microbial ecology quantitative analysis software version 1.9.0.

Sample size and statistical analysis

In our pilot study, the incidence of post-operative neurological dysfunction in the probiotic group was 6.3% and in the control group 22.2%. Significance was set to 0.05, power was set to 80%, and the sample size required to detect the difference was 120 cases according to the formula:

wherein, delta is (p1-p2), p is (p1 and p2)/2, and p1 and p2 are the positive rates of the experimental group and the control group respectively. Assuming an out-of-visit rate of about 20%, 150 patients were therefore included in the study.

Baseline characteristics were summarized using standard descriptive statistical methods. Normal distribution of data was determined using Kolmogorov-Smirnov test or Shapiro Wilk test. The Pearson's χ 2 test was evaluated for the primary endpoint (incidence of postoperative cognitive dysfunction). Secondary results, plasma IL-6 and cortisol changes were tested by Mann-Whitney U, postoperative pain intensity and sleep quality by two-way repeated measures ANOVAs, and gastrointestinal function recovery and perioperative changes in fecal flora composition were tested by unpaired t-tests. SPSS18.0 software was used for all statistical analyses, and GraphPad 7.0 software was used for all plots. All statistical tests used a two-sided alpha value of 0.05. The ethical committee of xiangya hospital, central and south university, was responsible for supervising all data.

And (4) analyzing results:

demographic and perioperative variables

A total of 150 patients were recruited after evaluation of eligibility criteria during the period from 2018 month 1 to 2019 month 1. These 150 participants were equally randomly assigned to the probiotic group or control group (n-75 per group). However, 30 cases (day 14 in the probiotic 16 and control groups) were subsequently excluded from the study within 7 days, with exclusion factors including Dukes' stage D, colorectal perioperative cerebral infarction, additional non-operative treatments, or other factors (see fig. 1). The remaining 120 randomized patients (59 in the probiotic group and 61 in the control group) completed the study. The last visit time for the last randomized patient was 12 days 1 month 2019. A flow chart of the entry and random process is shown in fig. 1. No side effects associated with probiotic treatment occurred in the patients. Baseline characteristics were well matched between groups, see table 1:

TABLE 1 patient Baseline characteristics

All data are expressed as numerical percentages or mean values (SD).

Post-operative neurological dysfunction

The mean MMSE score (25.19 ± 3.37vs.25.39 ± 3.54, P ═ 0.743) at admission for the probiotic and control groups was not statistically significant (see fig. 2A). The MMSE score of the probiotic group patients is obviously higher than that of the control group (0.92 +/-2.05 vs-0.05 +/-2.40, and P is 0.020) on the 7 th day after admission and operation. The incidence of post-operative cognitive function impairment was significantly lower in the probiotic group (3 of 59 patients [ 5.1% ]) than in the control group (10 of 61 patients [ 16.4% ]) (P ═ 0.046) (see fig. 2B).

Changes in plasma IL-6 and cortisol

Control group had slightly elevated plasma IL-6 levels on day1 post-surgery and declined on days 5-7 as compared to pre-surgery (assessed in the morning on the day of surgery). The decrease of the probiotic group at days 5-7 after the operation is obviously greater than that of the control group (-117.90 + -49.15 vs-14.93 + -15.21, P ═ 0.044) (see FIG. 3A). On days 5-7, the decrease in cortisol levels in the probiotic group was also significantly greater than in the control group (-158.70 ± 53.52vs.40.98 ± 72.48, P ═ 0.010) (see fig. 3B).

Postoperative pain intensity, postoperative sleep quality, gastrointestinal function recovery

Within 7 days post-surgery, the NRS scores for pain at rest and while exercising were gradually decreased, with no statistical difference (P >0.05) (see fig. 4A).

NRS subjective sleep quality score (see fig. 4B), gastrointestinal function recovery score (see table 2 below):

the difference between the two groups was not statistically significant (P > 0.05).

TABLE 2 intraoperative data, length of stay and antibiotic treatment

All data are expressed as numerical percentages or mean values (SD).

Perioperative changes in fecal flora

The effect of perioperative probiotic treatment on fecal flora was determined by gDNA analysis based on 16S rRNA amplicon sequencing (16S sequencing). The highest phyla of bacteria and the first 20 bacteria in the pre-treatment and post-operative stool specimens of the probiotic and control groups are shown in fig. 5. The relative perioperative abundance of the probiotic groups varied overall at the genus level more than the control group (fig. 5B), with insignificant differences at the phylum level (fig. 5A), as seen in the following table:

p value

it is understood that post-operative neurocognitive dysfunction is multifactorial and its development is due to a complex interaction between the patient's original susceptibility and acute causative factors. Thus, the complexity of this etiology greatly increases the difficulty of preventing and treating post-operative neurocognitive disorders. Although many pharmacological and non-pharmacological approaches have been reported to be effective for both prevention and treatment, the incidence of post-operative neurocognitive impairment remains high and new, simple and effective methods of prevention are needed. In this study, we found that perioperative probiotic treatment can significantly reduce the incidence of postoperative cognitive dysfunction in elderly non-cardiac surgery patients without significant side effects. Perioperative probiotic treatment also significantly reduced plasma IL-6 (plasma interleukin-6) and cortisol levels. Interestingly, previous studies have shown that blood IL-6 levels are negatively correlated with the occurrence of post-operative neurocognitive dysfunction. Perioperative stress can exacerbate the impairment of post-operative neurocognitive function. Thus, in this study, perioperative probiotic treatment may significantly alleviate post-operative cognitive impairment in elderly patients by limiting peripheral inflammation and stress responses.

There are many methods for cognitive assessment, such as neuropsychological testing, MMSE, and montreal cognitive assessment scales. Among these tests, neuropsychological tests are generally recommended, but this takes about 2.5 hours, limiting its use in surgical wards. In contrast, MMSE is the most widely accepted screening tool to track decline in longitudinal cognitive function, typically requiring a time of around 10 minutes. MMSE is reliable in tracking down longitudinal cognitive function, although it is often criticized as less sensitive in diagnosing specific cognitive disorders. Previous studies have shown that the change in MMSE score in alzheimer's patients is typically less than 3 minutes per year. A significant decline in cognitive performance is generally defined as a change in MMSE score of 3 points or more. Therefore, to increase the reliability of our data, we follow the standards used in the prior art.

In terms of safety, most studies found that there was no risk of probiotic treatment after selective surgery. Also, in our study, perioperative probiotic treatment did not cause any significant side effects. The probiotic group and the control group have no difference in postoperative complication parameters such as blood loss, hospitalization time, gastrointestinal function recovery, antibiotic treatment and the like, and the probiotic treatment is safe to non-critical patients in the perioperative period. Of course, further large-scale studies are needed to rule out potential safety hazards that may exist with perioperative use of probiotics.

Another problem is whether the effect of perioperative probiotic treatment is affected by antibiotics, which are often used to prevent or treat surgical infections. In our study, we did not limit the use of postoperative antibiotics in order to avoid affecting the surgeon's clinical work, although there was no difference in antibiotic use between the final two experimental groups. Despite the use of antibiotics, we have observed that perioperative probiotic treatment has a significant protective effect on post-operative cognitive dysfunction. Similarly, previous studies have also reported the prevention of postoperative complications of infection by probiotic treatment in the presence of antibiotics. Thus, short term use of antibiotics (several days) is not an obstacle to perioperative use of probiotics. Of course, the protective effect of perioperative probiotic treatment on post-operative neurological dysfunction may be enhanced in the absence of antibiotics.

In summary, our data show that perioperative probiotic treatment can reduce cognitive impairment in elderly patients after non-cardiac surgery. Thus, probiotics may be a safe, inexpensive, and effective method of preventing post-operative cognitive dysfunction.

TABLE 3 postoperative recovery of gastrointestinal function

All data are expressed as mean (SD).

It will be appreciated that the probiotic combination, i.e. the strain selected from the group consisting of bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis, described in the present disclosure, has a preventive and therapeutic effect on post-operative neurological disorders in patients with non-cardiac surgery in the elderly in a general sense.

More preferably, the viable count of Bifidobacterium longum, Lactobacillus acidophilus and enterococcus faecalis is not less than 1.0 × 10⁷CFU。

The following are experiments by feeding probiotic bacteria to mice:

6-week-old C57BL/6J mice were selected and randomized into 4 groups. The control group (group C), the probiotic group (group CP), the stress group (group S), and the stress + probiotic group (group SP) were used. Wherein:

control group (group C): normally breeding for 6 weeks, and freely eating and drinking water;

probiotic group (CP group): feeding with probiotic (Bifidobacterium triple powder including Bifidobacterium, Lactobacillus, and enterococcus) daily, replacing drinking water and probiotic daily, and ensuring that the daily consumption of probiotic is more than 10 for each mouse⁷；

Stress group (S group): feeding the chickens in the first two weeks normally, drinking water by free diet, then carrying out constraint stress for 4 weeks, wherein the chickens cannot drink water by diet when stressed, and the chickens stop drinking water by free diet when stressed;

stress + probiotic group (SP group): feeding with probiotic (Bifidobacterium triple powder) daily, changing drinking water and probiotic daily, and administering drinking water daily in an amount equivalent to drinking amount to ensure that the daily consumption of probiotic is more than 10 for each mouse⁷(ii) a In the first two weeks, except for adding probiotics into drinking water, the animals are normally raised, and then are subjected to constraint stress of 4 weeks in behavior period (the animals are continuously fed with probiotics in the two weeks), so that the animals cannot eat or drink water during stress, and stop eating or drinking water freely during stress.

The four groups started to conduct the behavioural test in the evening of the day after the last stress of the stress group. The order of the behavioural tests was: open field experiment, new object identification experiment, Barnes maze experiment, social interaction experiment and forced swimming experiment. As shown in the flow chart of the behavioral experiment of fig. 6-1.

In FIG. 6-1, restraint stress was performed for 8-10 weeks. Day of stress completion was recorded as day 1. Day1 was conducted as an open field experiment, Day2-Day3 was conducted as a new object identification experiment, Day4-Day7 was conducted as a Barnes maze experiment, Day8 was conducted as a social interaction experiment, and Day9 was conducted as a forced swimming experiment.

Chronic stress: mice were restrained in perforated 50ml centrifuge tubes at 6 pm-9 am each day. Since the centrifuge tube is comparable in size to the mouse, the mouse is restricted from movement during this period and is prohibited from eating or drinking. Mice were released to free movement at 9 points each morning. The side wall, the front wall and the rear wall of the centrifugal tube are perforated for the mouse to breathe. To allow uniform treatment of individual mice, 49 wells were punched in each centrifuge tube. After daily release of the mice, the centrifuge tubes, especially the clogged holes, were carefully cleaned of fecal material.

Open field experiment

In a box with the length, width and height of 50 cm, 50 cm and 38 cm respectively, the bottom surface of the box is divided into 25 square small grids with the length of 10 cm and the width of 10 cm, wherein the middle 9 grids are defined as a central grid. The ability of the mice to freely explore was tested to reflect the activity of the mice. The experimental times were all started at 6 pm. The light of the action room is adjusted to be soft and darker, and the mouse can be seen clearly by the camera. The rats are moved to a behavior room to adapt to the environment at least half an hour in advance. The behavior room is kept clean and quiet without noise interference. The temperature and the humidity are both in a comfortable range, so that additional stimulation to the mice is avoided. During the experiment, a skilled animal experimenter puts the head of each mouse into the middle grid of the open field box in the same direction, and uses a camera to record video to observe the activity condition of the mouse in the open field within 5 minutes. The total number of boxes the mice walked in the open field box for 5 minutes was recorded, as well as the percentage of central boxes/total boxes. The number of the walking grids is based on the standard that the four limbs of the mouse cross the grids. Between every two rats, the excrement of the rats in the box is carefully cleaned, and the bottom and the frame of the open field box are wiped with 75% alcohol to remove the residual smell of the previous rat. After placing the mice in the open field box, the operator should leave the behavior room immediately.

Experiment of new object recognition

In a box with the length, width and height of 30 cm, 30 cm and 38 cm respectively, the preference of the mouse on the novel object is tested by utilizing the curiosity of the mouse on the novel object so as to reflect the memory capability of the mouse. The experimental times were all started at 6 pm. The light of the action room is adjusted to be soft and darker, and the mouse can be seen clearly by the camera. The rats are moved to a behavior room to adapt to the environment at least half an hour in advance. The behavior room is kept clean and quiet without noise interference. The temperature and the humidity are both in a comfortable range, so that additional stimulation to the mice is avoided. The experiment was divided into two phases, the first phase being the familiarity phase: two identical objects are placed at the bottom of the box along the diagonal line, the two objects are symmetrically placed, and the distance from the two objects to the box walls on the two sides is completely consistent. The mouse head was released towards the other diagonal by a skilled animal experimenter, and the distance of the mouse from the two objects was equal. After mouse release, two objects were freely explored in the box for ten minutes. The second stage experiment was performed after 24 hours. The second stage is a test period: and two objects are placed on the bottom of the box along the diagonal line at the same placing position as the familiar period, wherein one object is the same as the familiar period, and the other object is changed into an object with obviously different shapes and styles. The two objects are symmetrically placed and have the same distance from the box walls on the two sides. The mouse head was released towards the other diagonal by a skilled animal experimenter, and the distance of the mouse from the two objects was equal. After release, mice were freely explored in the box for ten minutes and the behavior of the mice was recorded with a camera. Between every two rats, the excrement of the rats in the box is carefully cleaned, and the bottom, the frame and the objects in the box are wiped by 75% alcohol to remove the residual smell of the previous rat. After placing the mouse in the box, the operator should leave the behavior room immediately. The objects used in the experiment, one was a flat cell culture bottle and one was a cylindrical coffee pop-top can, both were of equal height and size, but the shapes and colors were different and had sufficient weight that the mouse could not push. After the experiment was completed, the time taken for the mouse to search for the old object and the new object, respectively, was counted, and a new object search Preference index (Preference index) was calculated. The preference index is new object time/(new object time + old object time). The higher the discrimination index, the better the memory of the mouse for the old.

Barnes maze experiment

The Baens maze tests the space learning and memory ability of a mouse by utilizing the temperament that the mouse is afraid of light, likes darkness and likes exploration. The bayns maze is mainly composed of a circular platform 140 cm away from the ground. The diameter of the circular platform is 122 cm, the periphery of the platform is provided with 20 holes, and the diameter of each hole is 5 cm. The lower part of one hole is connected with the safety box, the lower parts of the other holes are all suspended, the safety box cannot be seen from the surface of the platform, and the safety box can be found only when the safety box reaches the corresponding hole. A 200 watt light bulb was suspended approximately 20 cm directly above the middle of the platform. Before the experiment is started, the rats are moved to a behavior room for adaptation to the environment at least half an hour in advance. The behavior room is kept clean and quiet without noise interference. The temperature and the humidity are both in a comfortable range, so that additional stimulation to the mice is avoided. The start time of the experiment was selected to start at 8 am. The behavior room light is in a soft, darker state. Before the experiment, the 200W headlight is turned off, the mouse is firstly placed in the black plastic cylinder in the middle of the maze for 15 seconds, the headlight is turned on after 15 seconds, the black plastic cylinder is taken away, the mouse is guided to enter the safety box, and then the headlight is turned off to stay in the safety box for 1 minute. After 15 minutes, the mouse is placed in the black plastic cylinder in the middle of the maze again for 15 seconds, after 15 seconds, the headlight is turned on, the black plastic cylinder is taken away, the mouse can freely explore on the platform, the time for finding the safety box on the platform is recorded, and the number of times of finding wrong holes (wrong number of times) before finding the safety box is recorded. After entering the safety box, the rats were allowed to stay in the safety box for 1 minute. If the mouse does not find the safety box within 3 minutes, the mouse is guided into the safety box again, the time is recorded as 3 minutes, and the number of times of making mistakes is recorded according to the number of times of making mistakes. After introduction into the safety box, it was left in the safety box for 1 minute. In the process of searching the safety box by the mouse, one mistake is calculated by taking the condition that the horizontal line of the platform is detected by two eyes as a standard. Each mouse was tested three times per day with 15 minute intervals between each two, for 4 consecutive days. The experimenters should wear the same experimental clothes every day and wear the mask and the cap. When the mouse is explored on the platform, the experimenter should be at least 1 meter away from the platform and fix the position. Since the taste left by the previous mouse had a greater effect on the next mouse's exploration behavior, the excrements of the mice on the platform were carefully cleaned and the platform and all wells were wiped with 75% ethanol at each experimental interval to eliminate the odor effect.

Social interaction experiment

The experiment utilizes the characteristic that mice like to live in a social group to carry out a three-chamber social interaction experiment. The three-chamber social contact box is composed of three rectangular transparent resin glasses. The total length is 60 cm, width is 40 cm and height is 40 cm. Each box is 20 cm long, 40 cm wide and 40 cm high. Two clapboards for separating the three boxes are provided with a small hole of 5 cm by 5 cm at the lowest part of the middle part, so that rats can conveniently freely shuttle in each box. Two boxes at two ends are symmetrically provided with an inverted metal cage, the side wall of the metal cage is reticular, and the inside of the metal cage can be seen from the outside. The start time of the experiment was 9 am. The light of the action room is adjusted to be soft and darker, and the mouse can be seen clearly by the camera. The rats are moved to a behavior room to adapt to the environment at least half an hour in advance. The behavior room is kept clean and quiet without noise interference. The temperature and the humidity are both in a comfortable range, so that additional stimulation to the mice is avoided. The experiment was divided into two phases, the first phase, the familiarity phase: in the social communication box, the inverted metal cages are placed in the boxes at two ends, but no mouse is placed in the boxes, and the test mouse is released from the middle box and freely explores the three boxes for 5 minutes. Second stage, test period: a strange mouse is placed in the metal cage with one end of the box reversely buckled, the rest is the same as the first stage, the test mouse is released from the middle box again, the three boxes are freely explored for 10 minutes, and the behavior of the mouse in the boxes is recorded by a camera. And later-period analysis video records the stay time of the test mouse in the strange mouse box. After the mice were placed in the social interaction box, the operator should leave the behavior room immediately. Between each experiment, the excrement of the mice in the cage was carefully cleaned, and the social interaction cage and the metal cage were wiped with 75% ethanol to eliminate the influence of the residual smell of the previous mouse.

Forced swimming experiment

Forced swimming experiments are the classic way to conduct a research on depressive behaviour. The sex of a rat who is afraid of water but naturally swims is utilized. When the mouse is placed in a closed water environment, the mouse instinctively struggles to escape. When finding that the patient can not avoid, the patient gives up struggling and is represented as 'immobility', namely 'behavior desperate state'. The start of the experiment was 9 am. The rats are moved to a behavior room to adapt to the environment at least half an hour in advance. The behavior room is kept clean and quiet without noise interference. The temperature and the humidity are both in a comfortable range, so that additional stimulation to the mice is avoided. In the experiment, a transparent resin glass barrel with the diameter of 20 cm is used, water with the height of 20 cm is injected into the barrel, the water temperature is adjusted to be 22 +/-1 ℃, the mouse is placed into the forced swimming barrel, and the activity state of the mouse in the water is shot from the side by using a camera. Mice struggle vigorously for the first two minutes, with milder struggle behind. Post analysis video statistics the time 4 minutes after the mice were in the forced swim bucket to desperate. And (3) between every two experiments, replacing the water in the forced swimming barrel and cleaning excrement in the forced swimming barrel. After placing the mice into the forced swim bucket, the experimenter should leave the behavior room immediately.

Bacterial colony transplantation mouse experimental procedure

The schematic flow chart of the colony-transplanted rat experiment is shown in fig. 6-2, and 8-week-old male rats were randomly divided into 4 groups, namely, a control group (C), a stress group (S), a transplant group (F), and a sham transplant group (H). Wherein the control group is normally bred, the stress group is subjected to chronic constraint stress for 4 weeks (18 o 'clock-9 o' clock in the next day), the transplant group is subjected to flora transplant on 5 th, 12 th, 19 th and 26 th days of stress of the stress group, and the pseudo transplant group is performed on the same day as the transplant group. Behavioral testing was performed on day 28.

The behavioral results of the above experiments were analyzed as follows:

1. referring to fig. 7, in which,

c: control, C + P: control + probiotic group, S: stress group, S + P: stress + probiotic group

Fig. 7A shows that there was no significant difference in distance traveled by each group for 5 minutes in the open field experiments (F (3,63) ═ 0.9661, P ═ 0.4144);

FIG. 7B shows that in the new object identification experiment, the new object exploration rate of the stress group (group S) was significantly lower than that of the other three groups (S vs.C: P <0.0001, S vs.C + P: P <0.0001, S vs.S + P: P < 0.0001);

figure 7C shows that there was no significant difference in latency between groups of mice in the baens maze (F (3,78) ═ 2.059, P ═ 0.1125); repeated measures of anova showed significant differences between groups in the number of mistakes made (F (3,77) ═ 8.786, P < 0.0001); wherein the number of mistakes made by the group S is obviously more than that made by the other three groups (S vs.C: P is 0.0050, S vs.C + P: P is 0.0030, S vs.S + P: P is 0.0007);

figure 7D shows that in social interaction experiments, the S group showed lower propensity for social interaction than the C, C + P and S + P groups (S vs.c: P ═ 0.0010, S vs.c + P: P ═ 0.0375, S vs.s + P: P ═ 0.0084);

fig. 7E shows that immobility time was significantly increased in the S group compared to the other three groups in the forced swim test (S vs.c: P: 0.0323, S vs.c + P: 0.0018, S vs.s + P: 0.0277).

2. With reference to figure 8 of the drawings,

c: control, S: stress group, F: transplant group, H: pseudo-transplant group (only pipe, not transplant feces)

Fig. 8A shows that there was no significant difference in distance traveled by each group for 5 minutes in the open field experiments (F (3,42) ═ 0.9580, p ═ 0.4215);

fig. 8B shows that in the new object identification experiment, the new object search rate was significantly reduced in the S group compared to the C group and the H group (S vs.c: p <0.0001, S vs.h: p ═ 0.0376), and the new object search rate was also significantly reduced in the F group compared to the C group (p ═ 0.0011), while the S group was not significantly different from the F group (p ═ 0.8637);

figure 8C shows that in the barns maze experiment, repeated measures of anova showed significant differences between groups in comparison of latencies (F (3,42) ═ 8.459, P ═ 0.0002); latency was significantly increased in both S (p 0.0325) and F (p 0.0492) compared to group C, whereas S was not significantly different from group F (p 0.9885); repeated measures of anova showed significant differences between groups in the number of mistakes made (F (3,42) ═ 19.31, P < 0.0001); compared with the group C and the group H, the number of mistakes made by the group S is obviously increased (S vs. C: p is 0.0011, S vs. H: p is 0.0243); compared with the group C, the number of mistakes made in the group F was also significantly increased (p ═ 0.0133), and the group S was not significantly different from the group F (p ═ 0.7373).

It should be noted that the clinical data of the present application has been submitted to a third party for inquiry, registration number ChiCTR1800014365, detailed in the website:

http://www.chictr.org.cn/showproj.aspxproj＝22022。

as described above, the above embodiments are only used to illustrate the technical solutions of the present disclosure, and not to limit the same; although the present disclosure has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present disclosure.

Claims (2)

1. Use of pefeikang in the preparation of a medicament for preventing and/or treating postoperative neurological dysfunction in an elderly non-cardiac surgery patient, wherein the pefeikang comprises bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis.

2. The use according to claim 1, wherein the viable count of Bifidobacterium longum, Lactobacillus acidophilus and enterococcus faecalis is not less than 1.0 x 10⁷CFU。

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