CN114875018B

CN114875018B - Application of graphene irradiation in improving insulin activity in vitro

Info

Publication number: CN114875018B
Application number: CN202210509439.1A
Authority: CN
Inventors: 张倩; 胡格; 张蒙; 穆祥; 冯波; 张依; 冯晨; 鲁冠杰
Original assignee: Beijing University of Agriculture
Current assignee: Beijing University of Agriculture
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-11-21
Anticipated expiration: 2042-05-11
Also published as: CN114875018A

Abstract

The invention provides application of graphene irradiation in improving insulin activity in vitro.

Description

Application of graphene irradiation in improving insulin activity in vitro

Technical Field

The invention relates to application of graphene irradiation in improving insulin activity in vitro, and belongs to the technical field of diabetes treatment.

Background

Early animal experiments found that the serum, skeletal muscle, cardiac muscle, liver and fat levels of insulin in T2DM rats were significantly higher than those in normal rats, but blood glucose was still at higher levels, and this was the cause of this phenomenon, except for what was currently thought to be insulin resistance, we hypothesized that it is possible that the insulin activity was altered.

Prior studies have demonstrated that MVECs are both cells directly damaged by hyperglycemia and the primary regulator of insulin entry into tissues. Most previous studies were conducted around the effects of hyperglycemia on MVECs function. However, the interaction between insulin and MVECs in T2DM is still not completely understood. The insulin in blood possibly has the activity improved after passing through MVECs through the action of graphene, so that the relationship between insulin and MVECs is revealed as a direction for elucidating the pathogenesis of T2DM, and a scientific basis can be provided for the development of new medicines and new treatment means. First, insulin in the blood must pass through the walls of the micro-blood vessels to be delivered into the tissue. Secondly, MVECs basically express proteins such as insulin receptor (INSR), insulin-like growth factor-1receptor (insulin-like growth factor-1 receptor), glucose transporter (glucose transporters, GLUT) and the like, and provide a foundation for MVECs to directly participate in metabolism of insulin and sugar. Furthermore, it is speculated by the scholars that MVECs dysfunction may alter the trans-microvascular channel of insulin, causing abnormal glucose metabolism and thus insulin resistance. In view of this, we studied the influence of graphene on skeletal muscle microvascular endothelial cell structure and function by constructing skeletal muscle microvascular endothelial cell T2DM injury model, irradiating it with a graphene device, and determining the content of ET-1 and eNOS.

It should be noted that the foregoing description of the technical background is only for the purpose of facilitating a clear and complete description of the technical solutions of the present specification and for the convenience of understanding by those skilled in the art. The above-described solutions are not considered to be known to the person skilled in the art simply because they are set forth in the background section of the present description.

Disclosure of Invention

The invention aims to solve the technical problem that the prior art has the defects, and provides application of graphene irradiation in treating type 2 diabetes mellitus.

The scheme is realized by the following technical measures:

use of graphene irradiation for improving insulin activity in vitro.

Preferably, the graphene irradiation temperature is 39 ℃.

Preferably, the graphene irradiation time period is 30min. The invention has the beneficial effects that:

the invention can finely adjust the electrolytic steel plate and ensure the assembly aesthetic property.

Drawings

FIG. 1 primary cell migration;

FIG. 2 morphology of SMMVECs after purification;

FIG. 3 SMMVECs immunofluorescence assay;

FIG. 4 effect of different concentrations of PA on SMMVECs cell viability;

FIG. 5 effect of graphene time of action on SMMVECs cell viability;

FIG. 6 effect of graphene on SMMVECs secretion of ET-1 and eNOS;

FIG. 7 effect of graphene on time to serum insulin passage through endothelial cells;

FIG. 8 sets of insulin levels;

FIG. 9L 6 cell glucose uptake capacity of each group;

Detailed Description

In order to clearly illustrate the technical characteristics of the present solution, the present solution is described below by means of specific embodiments and with reference to the accompanying drawings.

1 test materials

1.1 selection of cells

Skeletal muscle microvascular endothelial cells (SMMVECs) were extracted, purified, and identified by individuals; l6 cells were purchased from luxuriant organisms.

1.2 major reagents

1.3 major instrumentation

2 test method

2.1 Cultivation of SMMVECs

2.1.1 Extraction of SMMVECs

The method for separating cells in skeletal muscle by adopting the patch method comprises the following specific operation steps:

(1) Removing neck, killing SD rat of 3 days old, performing aseptic dissection in an ultra-clean workbench, pouring excessive blood into ice-bath PBS (phosphate buffer solution) heart, cutting skeletal muscle of leg into precooled PBS, tearing into small blocks, and washing in new PBS for 3 times;

(2) Spreading the chopped skeletal muscle small blocks on sterile dry filter paper, carefully removing the muscle fibers, then putting the crushed skeletal muscle small blocks into new PBS, and washing for 3 times;

(3) Placing the tissue blocks washed by PBS in a 1.5mL centrifuge tube, and adding 0.5mL of serum;

(4) Cutting the tissue block into pieces with the diameter of about 1mm, and then adding 0.5mL of serum for uniform mixing;

(5) The liquid transfer device sucks the sheared tissue blocks, and vertically pumps the sheared tissue blocks into a 6-hole cell culture plate which is rinsed for 3 times in advance by PBS;

(6) Discarding the redundant serum, and obliquely placing a 6-hole cell culture plate inoculated with the tissue block into an incubator for about 20min;

(7) Excess serum was removed, 2ml of 10% ecm medium was slowly added to each well and placed in a cell incubator for 24h;

(8) After 24h, the medium was discarded, and after 3 times of washing with pre-warmed PBS, 10% ECM medium was added for further incubation for 36h;

(9) After 36h, observing that a large number of cells are dissociated to the bottom wall of the 6-hole cell culture plate under a microscope, and gently blowing to remove the tissue blocks;

(10) After 3 washes with warmed PBS, 10% ecm medium was added for continued culture until the cells of each well were completely confluent.

2.1.2 Purification of SMMVECs

Skeletal muscle tissue patches were cultured until a large number of cells were dissociated to the bottom wall of a 6-well cell culture plate (about 60 h), and magnetic bead sorting was performed from the cultured cells using CD31 molecules as specific markers in order to obtain endothelial cells of high purity. The specific operation steps are as follows:

(1) mu.L of the beads were aspirated, and the beads were rinsed 3 times with PBS solution (buffer 1) containing 0.1% BSA;

(2) 10. Mu.L of CD31 primary antibody and 40. Mu.L of buffer 1 were added;

(3) Placing a 1.5mL centrifuge tube on a shaking table, and incubating for about 40min at room temperature under shaking;

(4) Place 1.5mL centrifuge tube on pole for 1min, carefully discard supernatant;

(5) Adding 1mL of buffer solution for 1 washing 3 times;

(6) Same as in (4);

(7) 1mL of the cell suspension (about 1X 107) was added and mixed well, and incubated on a shaking table at 4℃for 20min;

(8) 1mL of PBS solution (buffer 2) containing 0.1% BSA and 2mM EDTA was added to prevent trapping of cells not bound to the magnetic beads;

(9) Place 1.5mL centrifuge tube on pole for 2min, carefully discard supernatant;

(10) Separating 1.5mL centrifuge tube from the magnetic pole, adding 1mL buffer solution 1, and washing for 2 times;

(11) Same as operation (9);

(12) After pipetting the supernatant, 200 μl of pre-warmed ECM solution containing 1% fbs, 1mm CaCl2 and 5mm MgCl2 (buffer 3) was added to a 1.5mL centrifuge tube;

(13) Adding 10 mu L of release buffer (DNase I), and incubating for 15min at room temperature with slight shaking;

(14) A new 1.5mL centrifuge tube was pretreated with 500 μl of pre-warmed buffer 3 for 5min;

(15) Placing the original centrifuge tube on a magnetic pole for 2min, and transferring the supernatant into the centrifuge tube with 1.5mL pretreated in the step (14);

(16) Resuspension the beads with 500 μl of pre-warmed buffer 3, repeating step (15) once;

(17) The supernatant in (16) was inoculated into a 6-well cell culture plate, and after 4 hours of culture, 10% ECM medium was added for further culture.

2.1.3 Identification of SMMVECs

(1) Inoculating the cells screened by 2.1.2 into a glass bottom dish, and fixing the cells with precooled 75% alcohol at 4 ℃ for 20min when the cells are converged to 70-80%;

(2) Rinsing the fixed cells 3 times with pre-chilled PBS solution for about 5min each;

(3) Blocking for 20min by adding 3% BSA solution, adding factor VIII antibody, and incubating overnight at 4deg.C;

(4) Washing 3 times by precooled PBS solution, adding FITC-labeled secondary antibody, and incubating for 30min at 37 ℃;

(5) 5 mu L of DAPI is added 15min before the secondary antibody incubation is finished to dye the cell nuclei for 15min;

(6) After the dyeing is finished, the sample is washed 3 times by a precooled PBS solution, and is observed and photographed under a microscope.

2.1.4 Subculture of SMMVECs

When the purified skeletal muscle microvascular endothelial cells are converged to about 80% -90%, the original culture medium is sucked and removed, the cells are washed 3 times by using preheated PBS, 0.125% pancreatin is added, the digestion state of the cells is observed under a microscope, 10% ECM culture medium is added to stop digestion when rounding and falling off, cell suspension is collected to a 15mL centrifuge tube, centrifugation is carried out at 1200rpm for 5min, the supernatant is removed, and after the 10% ECM culture medium is added to be resuspended, the culture is carried out according to a ratio of 1:2, and the culture is carried out continuously in an incubator.

2.1.5 Cryopreservation of SMMVECs

After centrifugation according to step 2.1.4, the supernatant was discarded, 1mL of frozen stock solution (FBS: dmso=9:1) was added to resuspend the cells and transferred to a 2mL cryopreservation tube, and the cells were frozen in gradient.

2.2 Establishment of T2DM skeletal muscle microvascular endothelial cell injury model

SMMVECs grown to F2-F6 generation were recovered, and after cells were confluent to about 80%, 0.125% of the pancreatin digested cells and cell concentration was adjusted to 5X 104 cells/mL, 100. Mu.L per well was inoculated into 96-well cell culture plates, and after culturing for about 12 hours to cell attachment, the cells were randomly divided into 6 groups: control group (only 5% ecm), HG group (10% ecm+25 μm glucose), palmitic Acid (PA) concentration groups (10% ecm+25 μm glucose+25, 50, 100, 200 μm palmitic acid). Cell activity was measured by MTT assay: and (3) treating cells according to groups, wherein 6 compound holes are formed in each group, zero setting holes are formed at the same time, after culturing for 24 hours, 10 mu L of 5mg/mLMTT is added into each hole, after light-shielding culture is continued for 4 hours, the original culture medium is discarded, 100 mu L of DMSO is added into each hole, shaking and mixing are carried out, and OD value is detected under the condition that absorbance is 490 nm.

2.3 Effect of graphene on T2DM skeletal muscle microvascular endothelial cell injury

2.3.1 determination of optimal action time of graphene on T2DM skeletal muscle microvascular endothelial cell injury

After constructing a T2DM skeletal muscle microvascular endothelial cell injury model according to the cell injury model building method in 2.2, randomly dividing the cells into 5 groups: control group, graphene irradiation 15, 30, 60, 120min group (Control graphene temperature 39 ℃). The MTT assay measures cell activity, as well as 2.2.

2.3.2 Effect of graphene on SMMVECs secretion of ET-1 and eNOS

And inoculating the 3 rd generation SMMVECs into a 6-hole cell culture plate, dividing the cell culture plate into a Control group, a Model group and a Gra-I group after fusing the cells, treating the cells according to the grouping, collecting cell culture supernatants, and detecting according to ELISA kit instructions.

2.4 Effect of graphene on insulin after trans-endothelial transport

2.4.1 Effect of graphene on insulin transendothelial optimal time

SMMVECs grown to F2-F6 generation are inoculated into a Transwell upper chamber (24 holes), 100 mu L of normal culture medium (10% ECM) is added into a lower chamber, each group is divided into 2h, 4h and 6h groups, 3 compound holes are arranged in each group, ECM culture medium containing 10% rat serum is replaced into the upper chamber after endothelial cells are attached (about 12 h), serum-free culture medium is replaced into the lower chamber, graphene is irradiated for 30min and then is continuously cultured for 2h, 4h and 6h according to groups, 0.5mL of lower chamber culture medium is sucked into L6 cells starved for 4h, 0.5mL of serum-free ECM culture medium containing 100 mu M2-NBDG is added, after uniform mixing, the culture medium is removed, the cells are digested with precooled PBS for 3 times, and are collected into a flow tube, and the flow tube is detected by an upper machine.

2.4.2 Effect of graphene on insulin content after trans-endothelial transport

SMMVECs grown to F2-F6 generation were inoculated into a Transwell upper chamber (24 wells), 100. Mu.L of normal medium (10% ECM) was added to the lower chamber, and the cells were divided into a normal group (Control group), a Model group (Model group), and a graphene irradiation group (Gra-I group), each group was provided with 3 duplicate wells, after endothelial cells were attached (about 12 hours), the Control group upper chamber was replaced with ECM medium containing 10% normal rat serum, the Model group and Gra-I group upper chamber were replaced with ECM medium containing 10% T2DM rat serum, the lower chamber was replaced with serum-free ECM medium, and Gra-I group graphene was irradiated for 30 minutes, and cell culture supernatants in the lower chamber were collected after 4 hours of group culture were continued, and insulin content in the cell culture supernatants was detected by radioimmunoassay.

2.4.32-NBDG method for detecting influence of graphene on glucose uptake capacity of insulin L6 cells after trans-endothelial transport

The cell grouping and culturing are same as 2.4.2, after continuously culturing for 4 hours according to the grouping, 0.5mL of lower chamber culture medium is sucked into L6 cells starving for 4 hours, 0.5mL of serum-free ECM culture medium containing 100 mu M2-NBDG is added, after uniform mixing, the cells are cultured for 30 minutes in a dark place, the culture medium is discarded, the cells are washed 3 times by precooled PBS, pancreatin is used for digesting the cells, and the cells are collected into a flow tube and are detected by an upper machine.

3 results

3.1 Extraction, purification and identification of SMMVECs

3.1.1 Observation of the emigration of SMMVECs

After separating skeletal muscle tissue cells by a paste method, observing the migration condition of the cells in 60 hours, wherein the tissues are scattered around the tissue blocks to form small bubbles when the cells are 6 hours as shown in figure 1; at 12h, a small amount of cells are bounced around the tissue block and take on an irregular fusiform shape; over time, cells swim more and more, grow densely at 60h, and are paving stone-like.

3.1.2 Purification results of SMMVECs

As shown in fig. 2, after purification for 12 hours, the cells grow on the wall, and the morphology is perfect and is in an irregular spindle shape; after 24 hours, the cells are gradually increased to form monolayer proliferation, and after 48 hours, the cells are paved on the bottom of the six-hole cell culture plate.

3.1.3 Results of authentication of SMMVECs

The identified cells were observed under an inverted fluorescence microscope, as shown in FIG. 3, with the cells in a single-layered confluent state. After fluorescent staining with factor viii antibodies, a clear green positive reaction was seen around the nuclei (fig. 3B), indicating that the cultured cells were SMMVECs.

3.2 T2DM skeletal muscle microvascular endothelial cell injury model

As shown in fig. 4: compared with Control group, HG group cell viability was significantly increased (P < 0.01), HG+25μm PA group cell viability was significantly decreased (P < 0.05), and cell viability was gradually decreased with increasing PA concentration. The following is indicated: the simple high sugar not only has no damage to the skeletal muscle microvascular endothelial cells, but also can enhance the cell activity, and when the high sugar and the palmitic acid are combined, the 25 mu M glucose and the 25 mu M palmitic acid can reduce the cell activity, and when the 25 mu M glucose and the 50 mu M palmitic acid act, the cell activity is reduced to 50%, so that the concentration is half inhibition concentration (IC 50) of the skeletal muscle microvascular endothelial cells, and the concentration is selected for subsequent experiments.

3.3 Effect of graphene on T2DM skeletal muscle microvascular endothelial cell injury

3.3.1 optimal time of action of graphene on T2DM skeletal muscle microvascular endothelial cell injury

As shown in fig. 5, compared with the Control group, the graphene was irradiated for 15min, the cell viability was not significantly changed (P > 0.05), the graphene was irradiated for 30min, the cell viability was significantly enhanced (P < 0.05), and the cell viability was significantly reduced (P < 0.01) when the graphene was acted for 120 min. The above results illustrate: the optimal action time of graphene irradiation on the T2DM skeletal muscle microvascular endothelial cell injury model is 30min.

3.3.2 Effect of graphene on SMMVECs secretion of ET-1 and eNOS

As shown in FIG. 6, the Model group ET-1 content was significantly increased (P < 0.01) (FIG. 6A) and the eNOS content was significantly decreased (P < 0.01) (FIG. 6B) as compared with the Control group; whereas Gra-I group had significantly reduced ET-1 content (P < 0.01) (FIG. 6A) and significantly increased eNOS content (P < 0.01) (FIG. 6B) compared to the Model group. It is suggested that T2DM may cause SMMVECs dysfunction, while graphene is able to repair this functional impairment.

3.4 Effect of graphene on insulin after trans-endothelial transport

3.4.1 optimal time of graphene to insulin transendothelial

As shown in fig. 7, the glucose uptake capacity of L6 cells was strongest at 4h after graphene irradiation, the difference was significant compared to 2h (P < 0.01), and the glucose uptake capacity of L6 cells was significantly reduced at 6h after graphene irradiation compared to 4h after graphene irradiation (P < 0.01). The results show that serum insulin has the best effect of penetrating through endothelial cells after the graphene is irradiated for 4 hours, so that the following experiment selects the time for penetrating through skeletal muscle microvascular endothelial cells after the graphene is irradiated for 4 hours.

Effects of 3.4.2 graphene on insulin content after trans-endothelial transport

As shown in fig. 8, the Model group insulin was significantly increased (P < 0.01) compared to the Control group, and the Gra-I group insulin was significantly decreased (P < 0.01) compared to the Model group, indicating that graphene irradiation can reduce the amount of insulin after trans-endothelial transport and increase insulin sensitivity.

3.4.3 Effect of graphene on glucose uptake by insulin L6 cells after trans-endothelial transport

As shown in fig. 9, the Model group had significantly reduced glucose phagocytic capacity (P < 0.01) compared to the Control group; compared with the Model group, the Gra-I group has significantly enhanced glucose phagocytic capacity (P < 0.01), which indicates that graphene irradiation can improve the glucose uptake capacity of the L6 cells of insulin after trans-endothelial transport, and in combination with animal experiments, we speculate that: under the action of graphene, SMMVECs may affect the activity of insulin passing through, but specific mechanisms need further investigation.

The technical features not described in the present invention may be implemented by the prior art, and are not described herein. The present invention is not limited to the above-described embodiments, and variations, modifications, additions, or substitutions within the spirit and scope of the present invention will be within the scope of the present invention by those of ordinary skill in the art.

Claims

1. The application of graphene irradiation in improving insulin activity in vitro is characterized in that after the graphene is irradiated on skeletal muscle microvascular endothelial cells, the capacity of insulin transported across the endothelium for 4 hours for stimulating L6 cells to take up glucose is enhanced, the irradiation temperature is 39 ℃, and the irradiation time is 30min.