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Strain Stimulations with Different Intensities on Fibroblast Viability and Protein Expression

  • Ying Jia , Junmei Zhang , Bo Chen EMAIL logo , Minghong Luo , Weiyin Cheng , Yalin Wang , Juan Liu and Hua Yang
Published/Copyright: October 23, 2017
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Open Life Sciences
From the journal Open Life Sciences Volume 12 Issue 1

Abstract

Background

Mechanical stimulation via acupuncture and tuina massage triggers various cell responses. This study aims to understand these cellular bio-physical mechanisms by investigating the effect of different stimulation intensities on cell viability and protein expression.

Methodology

Connective tissue fibroblasts were cultured in vitro. Three varying intensities of mechanical strain stimulation were applied to the cells, either once or three times and compared with non-stimulated controls. Changes in fibroblast viability and fibroblast protein expression were observed.

Results

Strain stimulation intensity significantly increased fibroblast cell survival rate (p<0.01) to effectively improve cell viability. Moreover, the combined influence of both the strain stimulation intensity and number of stimulations on the fibroblast survival rate significantly differed (p<0.05). Strain intensity also significantly altered fibroblast protein expression between the three groups (p<0.0001). Cluster analysis showed that the medium-intensity strain stimulation posed the maximum influence on protein expression.

Conclusion

The difference in cell viability and protein expression of the connective tissue fibroblast during the in vitro strain process reveals the cytobiological mechanism of basic medicinal mechanical stimulation.

Keywords: Strain stimulation; connective tissue; fibroblast; cell viability; protein expression; cluster analysis

1 Introduction

Studies on acupuncture and moxibustion in modern Chinese traditional medicine and Western medicine demonstrate that different forms of mechanical stimulation cause varied biological behaviors in cells [1,2]. Specifically, various cells respond to mechanical stimulation by altering their functional status and generating different bioactive substances that have drug-like or hormone-like biotherapy effects [3,4]. Various disciplines focus on varied and specific forces and different types of stimulation [5]. However, the breakdown analysis of these forms of mechanical stimulation suggests that they are composed of several individual forces such as pressure, strain, and shear force (inner abrasion) [6,7].

Our prior studies indicated that connective tissue fibroblasts respond to pressure and strain (in an in vitro simulation model) via β1 type integrin. Pressure stimulation in particular promotes cell proliferation activities and increases the release of multiple bioactive substances such as NO, PGE2, MMP-1, TIMP-1, IL-1, and IL-6 [8-10]. Additionally, pressure stimulation down-regulates and/or maintains the synthesis of bioactive substances, such as IGF-1, which fulfills the potential regulation effects of functional proteins. In contrast, strain stimulation may effectively increase and facilitate the synthesis of total quantity of proteins within the fibroblast [11,12]. It remains unclear whether strain stimulation has any effect on fibroblast viability or protein expression within the cells. Currently, there are no clear answers to these questions.

Therefore, this study focused on connective tissue fibroblasts, as observation of differences in fibroblast viability and protein expression may help further understand the influence of strain stimulation on the biological behavior of fibroblasts. Thus, the common mechanism of cytobiological therapy induced by medicinal mechanical stimulation is explored.

2 Materials and Methods

2.1 Primary and passage culture of connective tissue fibroblasts

For the tests, we used pregnant (14th day of pregnancy) mice (Chongqing Tengxin Biotechnology Co., Ltd., Animal license No.: SCXK (Army) 2012-0011). The animals were sacrificed via cervical dislocation. Then, abdominal cavities were dissected, and uterus was cut open to expose the fetus. Each sacrificed animal was placed under the dissecting microscope, and the skin and subcutaneous tissue were obtained from the posterior midline (a 2 mm width from the neck to the lumbosacral area). A scalpel was used to scrape down fascial connective tissues and to remove fat and blood vessels and the resulting subcutaneous tissue was placed into a culture dish. Then, the tissue was cut using ophthalmic scissors to prepare a slurry, to which was added 3 ml of 0.1% I type collagenase. The general in vitro experiment protocol was followed to extract, cultivate, and amplify the fibroblasts.

2.2 Strain loading tests for connective tissue fibroblasts

A four-point flexible cell strain plate (Flexcell International Corporation, Chicago, USA) was immersed in concentrated sulfuric acid overnight. The plate was then washed with clean water and rinsed with triple-distilled water several times. Then, the plate was dipped into 75% alcohol for 24 h to remove possible fatty components. Following this, the plate was placed into an aseptic culture dish and moved to a super-clean bench for UV double-side sterilization. Then, the cells were inoculated. The 5th - 8th passage of the fascial connective tissue fibroblasts was used; 0.25% pancreatin + EDTA 0.5 ml was added to prepare the cell suspension. The cell density was adjusted to 1×104/ml for the inoculation on the four-point flexible cell strain plate. In total, 1 ml of cells was inoculated in the middle of each strain plate and cultivated in the incubator at 37°C, 5% CO2, and 100% humidity. After 2-4 hours, the strain plate was placed into the culture dish, and 20 ml of serum-free DMEM was added for another 24 hours of cultivation. Once the cells were synchronized, the solution was replaced with 20 ml of 10% FCS of DMEM substrate. The cells were randomized into four different groups according to the magnitude of strain force to be imposed, i.e. 0 μ strain, 1000 μ strain, 2000 μ strain, and 3000 μ strain, with 6 samples in each group. Cell viability index and cell diameter were measured by BECKMAN Cell Viability Analyzer (VICELL AUTO100 / 240) as indicated [13,14]. Briefly, fibroblast cells were digested with trypsin after stimulation, and then the fibroblasts digested in the cell suspension were detected for testing. In addition, during the cell viability study, each strain group was further randomized into two groups according to the number of forces applied, with one group receiving the stimulating force only once (single force subgroup) and the other receiving the stimulating force three times (multiple force subgroup) with three samples in each subgroup. The cells from each strain group were placed into a four-point flexible strain loading box, and then to a four-point flexible loading device. Then, the entire setup was placed into an incubator. The four-point flexible loading device was adjusted to the displacements of 0.5 mm (1000 μ strain), 1 mm (2000 μ strain), and 1.5 mm (3000 μ strain) with a force application frequency and time for each cycle set to 0.5 Hz and 2 hours, respectively. The interval of force application in the multiple force subgroup was 24 hours, and there was normal cultivation during the interval. After the force application, there was another 4 hours of cultivation. Then, the cells were collected for later inspection. The blank control group was placed into the incubator for regular cultivation without any stimulation and was inspected together with each force group.

2.3 Sample preparation and testing of the fibroblasts

The cells being exposed to the stimulating force were placed into the incubator for 4 hours of cultivation. The substrate was removed, and the cell culture plate was rinsed with PBS 3 times (5 min/cycle). The cell culture plate was gently agitated by adding a solution of 0.25% pancreatin + 200 μL EDTA, and the cells were spread evenly over the culture plate. The plate was placed into the incubator and digested for 5 min. A solution of 10% FBS containing 800 μL of DMEM was added to stop digestion, and the cells were again evenly spread in the culture plate. Then, the culture was moved into the measuring cup that was designed for viability testing. A viability instrument was used to test the cells.

2.4 Sample preparation and testing for fibroblast protein expression

The cells being exposed to stimulating force were placed into the incubator for 4 hours of cultivation. The substrate was removed, and the cells culture plate was rinsed with PBS 3 times (5 min/cycle). The cells culture plate was gently agitated by adding a solution of 0.25% pancreatin + 200 μL of EDTA, and then placed into the incubator and digested for 5 min. A solution of 10% FBS containing 800 μL of DMEM was added to stop digestion. Then, PBS solutions were used for repeated washing and for 3 cycles of centrifugation (1000 rmp, 8 min per cycle). Following this, the cells were collected and frozen for later inspection. Once the 6 samples were evenly mixed, AAM-BLM-1 antibody chip (supplied by RayBiotech, Georgia, USA) assays were performed (Supplemental Data Fig S1 and Table S1) according to the manufacturer instructions.

2.5 Real-time quantification PCR

Total RNA was isolated from cells using RNA isolation kit (Tiangen, Beijing, China) and reverse-transcribed into cDNA by M-MLV reverse transcriptase (Promega, Madison, WI). Real-time PCR was quantified by SYBR green mix (Takara, Dalian, China). Glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. The sequence of primers used are:

FAM3B, F, 5’-CCAGTTGCTACAGGCATCTTC-3’,

R, 5’-CGGTAGGCATAGGTGTCAGGA-3’;

CCR3, 5’-TGGCATGTGTAAGCTCCTCTC-3’,

5’-CCTGTCGATTGTCAGCAGGATTA-3’;

CCR6, 5’-TGGGCCATGCTCCCTAGAA-3’,

5’-GGTGAGGACAAAGAGTATGTCTG-3’;

IFN-gamma R1, 5’-GTGGAGCTTTGACGAGCACT-3’,

5’-ATTCCCAGCATACGACAGGGT-3’;

IL-9, 5’-ATGTTGGTGACATACATCCTTGC-3’,

5’-TGACGGTGGATCATCCTTCAG-3’;

uPAR, 5’-TGTGAGAGTAACCAGAGCTGC-3’,

5’-CCGAAGCACGGTAGTCCTG-3’; GAPDH,

5’-AGGTCGGTGTGAACGGATTTG-3’,

5’-GGGGTCGTTGATGGCAACA-3’.

2.6 Statistics

  1. Fibroblast viability study. All data were expressed as the mean ± standard deviation. The factorial ANOVA test from the SPSS19.0 statistics software was used to compare the variations between the test and control groups to determine if there was any statistical significance.

  2. Fibroblast protein expression study. The chemiluminescence imaging system was used to obtain exposure images (Clinx Science Instruments Co., Ltd, Shanghai, China). The raw signal value was obtained from the greyscale quantification, which was then corrected and standardized with a positive protein to acquire the standard data. From these standard data, we identified and counted the proteins that were changed greater than 2 times and lower than 0.5 times. The counts were analyzed using the SPSS19.0 software to perform the row × column c2 testing. This was done to compare cell protein expressions of each group under varying mechanical stimulation and to determine whether there was a statistical difference in inter-group changes. Finally, the clustering analysis chart was exported using the Treeview software to analyze the influence of varying mechanical stimulation on the cytobiological behavior of meridian-related fascial fibroblasts.

Ethical approval

The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

3 Results

3.1 Influence of in vitro strain stimulation on fibroblast viability

In Tables 1 and Table 2, the factorial ANOVA analysis suggested that in the single stimulation subgroup, the cell survival rate first increased and then declined as strain stimulation intensity increased. Specifically, the 2000 μ strain group was the highest in terms of the cell survival rate. A decrease in the cell survival rate was observed for the 3000 μ strain group, and the rate was lower than for the 0 μ strain group. However, in the multiple stimulation subgroups, the 1000 μ strain group had the highest cell survival rate. A decrease in the cell survival rate was observed for the 2000 μ strain and 3000 μ strain groups. Yet, the rates were higher than for the 0 μ strain group. Different stimulation intensities resulted in the fibroblast survival rate being significantly higher than in the blank control group (P<0.01). Number of stimulations did not significantly affect the fibroblast survival rate (P>0.05). A combination of pressure stimulation intensity and number of stimulations appeared to significantly affect the fibroblast survival rate (P<0.05).

Table 1

Influence of in vitro strain stimulations on fibroblast viability

Groups1 stimulation3 stimulationsTotal
nviability rate (%)nviability rate (%)nviability rate (%)
0 µstrain381.1667±1.44684380.5000±0.75498680.8333±1.09484
1000 µstrain383.9667±0.96090388.5000±0.79373686.2333±2.60512
2000 µstrain388.6333±1.92180385.5000±2.07846687.0667±2.48005
3000 µstrain380.6000±2.80000380.1667±4.06981680.3833±3.13332

Table 2

Tests of Between-Subjects Effects Dependent Variable: viability rate

SourceType III Sum of SquaresdfMean SquareFSig.Partial Eta Squared
Corrected Model268.203[(a)]738.3158.3670.0000.785
Intercept167852.1001167852.10036655.6000.0001.000
The times of stimulation0.03410.0340.0070.9330.000
The intensity of stimulation221.701373.90016.1380.0000.752
The times * the intensity46.468315.4893.3830.0440.388
Error73.267164.579
Total168193.57024
Corrected Total341.47023

3.2 Influence of in vitro strain stimulation on fibroblast diameter

In Table 3 and Table 4, the factorial ANOVA analysis suggested that in the single stimulation subgroup, the cell diameter first increased and then decreased during the strain stimulation intensity increase. Specifically, the 1000 μ strain group was the largest in terms of cell diameter. The cell diameter in the 2000 μ strain group was smaller than that of the 1000 μ strain group. The 3000 μ strain group had the smallest cell diameter, which was smaller than in the 0 μ strain group. In the multiple stimulations subgroup, higher strain stimulation intensity was associated with larger cell diameter. Specifically, the cell diameter was the largest in the 2000 μ strain group. Neither strain stimulation intensity nor number of stimulations had any significant effect on fibroblast diameter (P>0.05), nor did the combination of pressure stimulation intensity and number of stimulations (P>0.05).

Table 3

Influence of in vitro strain stimulations on fibroblast diameter

Groups1 stimulation3 stimulationsTotal
nCell diameter (µm)nCell diameter (µm)nCell diameter (µm)
0µstrain314.2733±0.14012314.3067±0.07767614.2900±0.10296
1000µstrain314.4567±0.17010314.3100±0.04359614.3833±0.13706
2000µstrain314.3867±0.03786314.4200±0.15716614.4033±0.10386
3000µstrain314.1000±0.23000314.3933±0.20984614.2467±0.25414

Table 4

Tests of Between-Subjects Effects Dependent Variable: Cell diameter

SourceType III Sum of SquaresdfMean SquareFSig.Partial Eta Squared
Corrected Model0.265[(a)]70.0381.6880.1820.425
Intercept4928.94714928.947219592.9530.0001.000
The times of stimulation0.01710.0170.7600.3960.045
The intensity of stimulation0.10130.0341.4940.2540.219
The times * the intensity0.14830.0492.1920.1290.291
Error0.359160.022
Total4929.57124
Corrected Total0.62423

3.3 Difference in the in vitro strain stimulation on fibroblast protein expression

A biotin-marked mouse antibody chip that is able to detect 308 proteins was chosen for this study. As shown in Fig. 1, positive control strains were visible, and negative control strains were not detected. The remaining proteins were irregularly presented. This suggests that the quality control was accurate, and the result was valid.

Figure 1 Difference in the in vitro strain stimulation on fibroblast protein expression A biotin-marked mouse antibody chip was chosen for this study, positive control strains (red box) were visible, and negative control strains (green box) were not detected. The remaining proteins were irregularly presented.
Figure 1

Difference in the in vitro strain stimulation on fibroblast protein expression A biotin-marked mouse antibody chip was chosen for this study, positive control strains (red box) were visible, and negative control strains (green box) were not detected. The remaining proteins were irregularly presented.

3.4 Influence of different strain stimulations on the difference in fibroblast protein expression

Up-regulated genes were defined as genes that had at least 2 fold increase in the test group compared with the control, as well as genes which were not expressed in the control but which were expressed in the test group. Stable proteins were defined as proteins with a positive expression in the blank control group and an up-regulation ratio of >0.5 and <2.0 in the test group. Down-regulated genes were genes positively detected in control, and at least fifty percent decreased in the test group. As shown in Table 5, strain stimulation induced clear regulatory effects on fibroblast protein expressions in all 3 groups with an inter-regulation ratio for the three groups of c2=75.817, P<0.0005. The regulation scale (the level of regulatory effect on protein expression, including both up-regulation and down-regulation) was highest for the 2000 μ strain group, which had the greatest quantity of up-regulated and down-regulated proteins. The regulation scale in the 1000 μ strain group remained between the values of the other two groups (Table 5, Supplemental Data Table S2 and Table S3).

Table 5

Difference in the in vitro strain stimulation on fibroblast protein expression

GroupsNumbers of upregulated proteinsNumber of non-regulated proteinsNumbers of downregulated proteinsTotal numbers
1000µstrain VS 0µstrain9415559308
2000µstrain VS 0µstrain9884126308
3000µstrain VS 0µstrain8317352308

3.5 Effect of varying strain stimulation on fibroblasts

To confirm the observed variation in cell protein expression, real-time quantification PCR was used to verify the chip screening. In all three strain groups the expression of FAM3B, CCR3, CCR6, and Pentraxin3 / TSG-14 was up-regulated, that is, significantly higher than in the control group and the expression of IFN-gamma R1, Chordin-Like 2, IL-9, and uPAR were down-regulated, significantly lower than in the control group. This may further confirm the chip results. (Fig. 2)

Figure 2 Influence of different strain stimulations on the difference in fibroblast protein expression Real-time quantification PCR was used to verify the chip screening to confirm the observed variation in cell protein expressions. In all 3 strain groups, we observed upregulated proteins FAM3B, CCR3 and CCR6; additionally, we also observed down-regulated proteins IFN-gamma R1, IL-9, and uPAR.
Figure 2

Influence of different strain stimulations on the difference in fibroblast protein expression Real-time quantification PCR was used to verify the chip screening to confirm the observed variation in cell protein expressions. In all 3 strain groups, we observed upregulated proteins FAM3B, CCR3 and CCR6; additionally, we also observed down-regulated proteins IFN-gamma R1, IL-9, and uPAR.

3.6 Cluster analysis of the influence of differing strain stimulation on fibroblast protein expression

The cluster analysis revealed a pattern of how differing strain stimulation may influence fibroblast protein expression. Specifically, 3000 μ strain had the lowest influence on the regulation of fibroblast protein expression, followed by 1000 μ strain. The 2000 μ strain had the maximum influence on protein expression and had the maximum variation in the cluster analysis (Fig. 3).

Figure 3 Clustering analysis of the influence of different strain stimulations on fibroblast protein expression The clustering analysis revealed a pattern of how different strain stimulations may influence the fibroblast protein expression. 3000 μ strain had the lowest influence on the regulation of fibroblast protein expression, followed by 1000 μ strain. The 2000 μ strain had the maximum influence on protein expression and had the maximum variation in the clustering analysis.
Figure 3

Clustering analysis of the influence of different strain stimulations on fibroblast protein expression The clustering analysis revealed a pattern of how different strain stimulations may influence the fibroblast protein expression. 3000 μ strain had the lowest influence on the regulation of fibroblast protein expression, followed by 1000 μ strain. The 2000 μ strain had the maximum influence on protein expression and had the maximum variation in the clustering analysis.

4 Discussion

Langevin et al showed that the anatomical relationship of acupuncture points and meridians to connective tissue planes is relevant to acupuncture’s mechanism of action and suggests a potentially important integrative role for interstitial connective tissue [15]. In the science of acupuncture and moxibustion, the mechanical analysis of filiform needle perforation shows that after a filiform needle is inserted into the acupoint, if the twisting action reaches a certain degree, the tissue cells in the acupuncture point region without a direct contact with the needle will be subjected to traction that propagates in the tangential direction of the twisting needle and is transferred by the cellular matrix that surrounds the needle. Moreover, if the needle swings during puncturing, the needle will induce traction stimulation on the surrounding tissues in the direction opposite to the needle’s swinging direction. Mechanical stimulation has also been studied in the area of orthodontics. In the biomechanical analysis of orthodontic appliances, the mechanical force exerted by the appliance on the teeth can be divided into stress and tension according to the mechanical effect. Pressure reduces the periodontal space, while tension increases the periodontal space. Increasing tension enhances the osteoblastic effect of fibroblasts within the normal load range of the periodontal ligament [16]. Thus is can be seen that strain effects are relevant to many areas of modern biomedicine and traditional medicine. The investigation of strain-induced cytobiological effects may provide a common guideline for explaining the therapeutic mechanisms of acupuncture and moxibustion in traditional Chinese medicine and Western medicine [17].

Cell viability refers to the physiological status and functions of the cell. Modern studies have shown that the mechanical factor is one of the major factors that affects cell viability. In this study, we used cell survival rate and cell diameter as cell viability indices. The results indicate that within a defined range of mechanical stimulation intensity and number of stimulations, strain stimulation may increase overall fibroblast viability and cell survival rate. Force intensity is the major factor that effectively increases the cell survival rate. Additionally, force intensity and the number of force stimulations may interact. Moreover, single (immediate effect) strain stimulation and multiple (accumulated effect) strain stimulations have different effects on cell survival rate. In single force stimulation, the cells are hypersensitive to medium force whilst in multiple force stimulation, the cells are hypersensitive to minor force. Within a particular range of force intensity and the number of force stimulations, strain stimulation may increase overall fibroblast growth (i.e., diameter increase). However, statistical analysis showed no statistical difference in the cell growth regardless of the individual factor of force intensity or number of stimulations or of the combination of both. Thus, strain may promote cell viability, but it mainly contributes to an increase in cell survival rate.

The changes in protein expression are closely related to changes in organic pathophysiology. Functional proteins are involved in many vital activities of living beings by delivering various specific physiologic functions, providing specific abilities to different cells and to the organism. For example, P53 could play an important role in somatic cells as “a guardian of the genome”; in addition, the new discovery of P53 in pluripotent stem cells have made possible significant progress in stem cell transplantation efficiency and safety [18]. The result of this study showed that strain stimulation induced regulatory effects on the expression of proteins from meridian-related fascial tissue fibroblasts in all three groups. The heavy strain group exhibited the lowest regulation (including up-regulation and down-regulation), and up-regulation prevailed. The maximum regulation was observed in the medium strain group, with the highest quantity of up-regulated and down-regulated proteins. The up-regulation and down-regulation in the minor strain group remained between the other two groups. Cluster analysis indicated the pattern of influence by different strain stimulations on fibroblast protein expression. Specifically, medium strain stimulation had the greatest influence on the expression of proteins with the maximum variation in the cluster analysis.

5 Conclusion

In conclusion, in vitro strain stimulation significantly altered cell viability and protein expression. In terms of cell mechanics, these changes may represent the cytobiological and therapeutic responses of fibroblasts to basic mechanical stimulation (pure strain stimulation). Medium-intensity strain stimulation may maximize cell viability and the regulation of protein synthesis. Although medium-intensity strain stimulation may significantly regulate cell protein synthesis, the down-regulated proteins increase significantly while the up-regulated proteins increase only slightly. This is a clear deviation from how low-intensity and high-intensity strain stimulations affect the cell, and warrants further investigation. Finally, after strain stimulation, the biomedical implications of up-regulation or down-regulation on cell proteins require further study and explanation.

Acknowledgment

This work was supported by Construction project of Key Laboratory of higher education in Guizhou Province (Guizhou Education KY [2014] No. 218); National Natural Science Foundation of China (No.81160456) and Guizhou Science and Technology Foundation (Guizhou Science Cooperation J [2014] No. 2030).

  1. Conflict of interest: Authors state no conflict of interest

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Received: 2017-5-3
Accepted: 2017-6-20
Published Online: 2017-10-23

© 2017 Ying Jia et al.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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https://doi.org/10.1515/biol-2017-0033
Keywords for this article
Strain stimulation; connective tissue; fibroblast; cell viability; protein expression; cluster analysis
Creative Commons
BY-NC-ND 4.0

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