Growth and Reproduction Characteristics of TLR4 Knockout Mice Used for Liver Fibrosis Experiments

Yue PENG Tiejian ZHAO Peng LIU Xuelian SUN Qing WANG

Abstract [Objectives] This study was conducted to investigate the similarity and differences between TLR4 knockout mice and C57BL/6 mice used in liver fibrosis research in terms of growth rate and reproduction ability. [Methods] Twenty TLR4 knockout mice and C57BL/6 mice， half male and half female， were selected to compare the growth rates of body weight and body length of mice from the 4th to 12th weeks; and 20 pairs of male and female mice of the same strain were compared for the number of baby mice of the second litter. [Results] The growth rates of body weight and body length of the TLR4 knockout mice were significantly lower than those of C57BL/6 mice （P<0.05） （except for the 4th and 5th weeks when there was no significant difference in body length）; and in terms of reproductive ability， the TLR4 knockout mice were significantly lower than the C57BL/6 mice （the ratio of the total number of baby mice in the second litter of the two strains， 72∶147）. [Conclusions] Knockout of the TLR4 gene has a significant impact on the growth and reproduction of mice.

Key words TLR4 gene knockout; Animal modeling; Liver fibrosis; Growth; Reproduction

Toll-like receptors （TLR） are a type of natural immune receptors， which are mainly distributed on the surface of immune cells. They can directly recognize and bind to certain pathogens or their products， trigger a series of signal transduction， and lead to the release of inflammatory mediators， thus playing an important role in natural immune defense[1]. Among them， TLR4 is the first TLR-related protein found in humans， and lipopolysaccharide （LPS）， the main component of the cell wall of Gram-negative bacteria， is its main ligand. Recent studies have found that TLR4 is expressed in various liver cells， and can regulate the natural immune response of liver cells， participate in the pathophysiological process of the liver， and play an important role in the occurrence and development of liver diseases[2-3]. The research community believes that hepatic stellate cells are the core cells for the occurrence and development of liver fibrosis[4]; hepatic stellate cells conduct signal transduction through activation of the lipopolysaccharide/TLR4 signaling pathway and directly promote the formation of liver fibrosis; and they directly enhance liver inflammation， affect HSC's own cell proliferation， apoptosis， collagen secretion and synthesis and fibrosis-promoting cytokine secretion through such two important signal pathways asTLR4/MyD88/NF-κB and MARKs， and promote the occurrence and formation of liver fibrosis[5]. Our research team conducted in-depth research on the pathogenesis of liver fibrosis. In order to eliminate the interference and influence of TLR4-related signal pathways， we purchased TLR4 knockout mice as the research model. In the ordinary feeding stage before the implementation of liver fibrosis animal modeling， our research team found that compared with C57BL/6 mice， the TLR4 knockout mice showed certain differences in growth rate and reproduction ability， so this paper provides complete and detailed comparative data， so as to provide a basis for the raising and reproduction techniques of the two kinds of mice and to provide a richer and more reliable experimental model basis for the experimental research of liver fibrosis based on the two kinds of mice.

Materials and Methods

Experimental animals

① Twenty clean-grade TLR4 knockout mice， half male and half female， 2 weeks old， weighing 7-10 g， purchased from Nanjing Biomedical Research Institute of Nanjing University， of strain C57BL/10ScNJNj， under the certificate number[SCXK（Su） 2015-0001].

② Twenty clean-grade C57BL/6 mice， half male and half female， 2 weeks old， weighing 10-15 g， purchased from Hunan SJA Laboratory Animal Co.， Ltd.， under the certificate number[SCXK（Xiang）2017-0001] and license number[SYXK （Gui）2015-0001].

During the experiment， human care was given in accordance with the 3R principle of experimental animals. The two mouse strains were raised according to the environmental requirements of clean animals. The environmental temperature was controlled at about 25℃， and the relative humidity was controlled at 30%-60%. The raising cages and drinking bottles were disinfected with 84 disinfectant and 75% ethanol every 3 d. The padding， feed， and water of the mouse cages were sterilized， and the padding was replaced every 3 d. The two mouse strains were raised and reproduced according one female and one male in the same cage in the whole process.

Main instruments

Electronic balance HLD-10001 （Sichuan Zhonglang Technology Co.， Ltd.）， with a maximum weighing weight of 1 000 g and an accuracy of 0.1 g; caliper A635051 （Shanghai Ace Electronic Technology Co.， Ltd.） with a range of 0-150 mm and a resolution of 0.05 mm.

Experimental methods

Every Monday， at the same time point， the weight and length of each group of mice were measured. Body length refers to the length from the midpoint of the connection between the ears to the junction of the tail and the body. When measuring the body length， a mouse was put on a stainless steel net， and when the mouse was grasping on the net， its tail was pulled to straighten it naturally （without applying excessive force， avoiding excessive stretching）， and then the length was measured. The growth rate was calculated according to Growth rate=（Body weight or length-Body weight or length in the second week）/Body weight or length in the 2nd week. The above data was measured from the 4th week to the end of the 12th week.

The breast-feeding period of mice is about 3 weeks， and they reach the stage of sexual maturity at 6 weeks of age. Therefore， mice from 6 to 12 weeks were selected for reproductive ability observation. The observation object was the number of baby mice of the second litter born by the male and female in the same cage， which could be used to represent the reproductive ability of the mouse[6-7].

Statistical methods

The data was statistically analyzed by SPSS22.0 software. The data of each group was expressed as the mean±standard deviation （x±s）. The homogeneity test of variance （F test） was performed on the obtained data. If the variances are uniform， the t test can be continued. If the variances are not uniform， the Tamhane' T2 method can be used. P<0.05 was regarded as significant difference.

Results and Analysis

Comparison of mouse growth

Comparison of body weight and weight growth rate between the two groups of mice

From the 4th week to the 12th week， in each week， the body weight and body weight growth rate of the TLR4 knockout mice were significantly lower than those of the C57BL/6 mice， and there were significant differences （P<0.05）. The details are shown in Table 1 and Fig. 1， Table 2 and Fig. 2.

Comparison of body length and body length growth rate between the two groups of mice

From the 4th week to the 12th week， the body length and body length growth rate of the TLR4 knockout mice were significantly lower than those of the C57BL/6 mice in each week， and there were significant differences in these two indexes （P<0.05）， （except that there were no significant differences in the 4th week and the 5th week， P>0.05）. The details are shown in Table 3 and Fig. 3， Table 4 and Fig. 4.

Comparison of mouse reproduction ability

The mice from the 6th week to the 12th week were selected to observe the reproduction ability. The TLR4 knockout mice and C57BL/6 mice were divided according to the same genus into cages， each included two （one male and one female）， and the number of baby mice of the second litter born in the same cage was counted to represent the reproductive ability of mice. The results showed that the reproductive ability of the TLR4 knockout mice was significantly lower than that of the C57BL/6 mice （the ratio of the total number of baby mice of the second parity， 72∶147）. The detailed data is shown in Table 5.

Conclusion and Discussion

Hepatic stellate cells are the core cells of liver fibrosis and the most critical cells that affect liver microcirculation[8]， and such two signaling pathways as TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs based on the action of hepatic stellate cells and mediated by TLR4 protein have a very important impact on liver inflammation， hepatic stellate cell proliferation and apoptosis， and the secretion level of fibrosis-promoting cytokine[9]. To study the pathogenesis of liver fibrosis， in-depth research based on TLR4-related signal pathways must be carried out， or the interference and influence of TLR4-related signal pathways must be excluded to facilitate the accurate conduct of liver fibrosis research. For the above two types of research， the TLR4 knockout mice have become a good research model with accurate target sites. However， the cost for modeling with the mice is high， and the mortality rate is also relatively high in the process of preparing animal models of liver fibrosis. Therefore， how to better obtain， culture， raise， and preserve the strain of mice is of great significance. In this study， it was found that the TLR4 knockout mice were significantly weaker than ordinary C57BL/6 mice in terms of body weight， body length growth rate， and reproductive ability （P<0.05）. The reason is very much related to the knockout of the TLR4 gene. The TLR4 gene is the initiating factor and key factor in multiple signal pathways， and mainly regulates the NF-κB and MAPKs signal pathways， and these signal pathways are important inflammatory pathways in the body. Gene TLR4 has been confirmed to be closely related to a variety of physiological immune phenomena and the occurrence and development of many diseases[10]. Studies have shown that TLR4 is closely related to the immune response process and the immunity of animals[11-12]. In the occurrence of inflammatory diseases， after using drugs to inhibit the expression of TLR4， it was found that the severity of inflammation and the level of immune response were significantly reduced[13]. After knocking out the TLR4 gene in experiments， there was no expression of TLR4 protein on the surface of immune cells， and the binding of TLR4 to pathogens was hindered， which would significantly affect the level of the animal's adaptive immune response[14]. We believe that the decline of immunity and physiological adaptation levels is an important reason why the knockout of TLR4 gene affects the growth and reproduction of mice.

During the feeding process of TLR4 knockout mice， we found that there was a significant infanticide phenomenon. Studies have shown that many knockout animals have infanticide phenomenon， which is significantly different from ordinary animals of the same strain[15]. Based on the study of Li et al.[16]， our research team improved the feeding method， optimized the observation and testing times on mice every day， and reduced the times of scares to mice， and we gave high-protein foods such as peanuts， sunflower seeds and cooked egg yolks to supplement the mice's nutrition and increase their feeling of fullness. It was found that the above two measures could alleviate infanticide phenomenon of mice. In addition， due to the decreased immunity and adaptability after TLR4 gene knockout， compared with C57BL/6 mice， the TLR4 gene knockout mice had higher environmental requirements. During the breeding process， if they were not disinfected in time or the sanitation of the breeding environment was improper， it could significantly increase the mortality rate of the knockout mice.

References

[1] KRISHNA SN. Liver fibrosis： a compilation on the biomarkers status and their significance during disease progression[J]. Future Sci. OA， 2017， 16（4）： 893-899.

[2] SHIBATA T， MOTOI Y， TANIMURA N， et al. Intracellular TLR4/MD-2 in macrophages senses Gram-negative bacteria and induces a unique set of LPS-dependent genes[J].Int Immunol， 2011， 23（8）： 503-510.

[3] PINZANI M. Foreword to liver fibrosis[J]. Best Pract Res Clin Gastroenterol， 2011， 25（2）： 193-194.

[4] CHENG Q， LI C， YANG SF. Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-β1/Smad and NOX4/ROS pathways[J]. Chemico-Biological Interactions， 2019， 2（2）：131-139.

[5] MAJUMDER S， PIGUET AC， DUFOUR JF. Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis[J]. Eur J Pharmacol， 2015， 75（1-3）： 86-95.

[6] TIAN XY， SUN JF. Study on the biological characteristics of inbred NJS mice： Observation of reproductive performance and growth and development[J]. Laboratory Animal Science and Administration， 1996（4）： 1-4. （in Chinese）

[7] LI ZD， WANG H， OUYANG TQ， et al. A comparative study on effects of three different illuminations durations on growth， development and reproduction of BALB/c Nude Mice[J]. Laboratory Animal Science， 2008（2）： 13-15. （in Chinese）

[8] SEKI E， BRENNER DA. Recent advancement of molecular mechanisms of liver fibrosis[J]. Hepatobiliary Pancreat Sci， 2016， 22 （7）： 512-518.

[9] GUO J， FRIEDMAN SL. Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis[J].Fibrogenesis Tissue Repair， 2010（3）： 21-24.

[10] ZHU XJ， ZHANG F， KONG DS， et al. Progress of Toll-like receptor 4 signaling transduction in hepatic fibrosis[J]. Chinese Journal of Pharmacology and Toxicology， 2013， 27（1）： 106-109. （in Chinese）

[11] YANG QG， LIU SN. P38MARK signal pathway and liver fibrosis[J]. World Chinese Journal of Digestology， 2012， 20（24）： 2231-2236. （in Chinese）

[12] YE L， CHEN ZY， YAN MX， et al. Activation and significance of MAPKs signaling pathway in liver tissue of rats with liver fibrosis[J]. Chinese Archives of Traditional Chinese Medicine， 2013， 31（12）： 2748-2750. （in Chinese）

[13] LIN XY， LIU JM. TLR4/MyD88/NF-κB signaling pathway and ulcerative colitis[J]. Chinese Journal of Gastroenterology， 2013， 18（4）： 244-245. （in Chinese）

[14] ZHU Q， ZOU L， JAGAVELU K， et al. Intestinal decontamination inhibits TLR4 dependent fibronectin-mediated cross-talk between stellate cells and endothelial cells in liver fibrosis in mice[J]. J Hepatol， 2012， 56（4）： 893-899.

[15] QIAO LX， XU M， CAI MY， et al. Breeding reproducing and identifying for p53 gene knockout mice[J]. Laboratory Animal Science， 2012， 4（2）： 100-102. （in Chinese）

[16] LI W， HE GY. Preliminary study of kronismus in Muc2 and DCN gene knockout mice[J]. Chinese Journal of Comparative Medicine， 2013， 23（4）： 28-30. （in Chinese）