Establishment and Evaluation of a Rat Model of lipopolysaccharide-high-fat diet Induced Sarcopenia

doi:10.21203/rs.3.rs-3416539/v1

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Research Article

Establishment and Evaluation of a Rat Model of lipopolysaccharide-high-fat diet Induced Sarcopenia

https://doi.org/10.21203/rs.3.rs-3416539/v1

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Objective

To establish and evaluate a rat sarcopenia model.

Methods

We divided 10-month-old male Sprague–Dawley (SD) rats into adult control (AC) and lipopolysaccharide-high-fat diet (LPS-HFD) groups, in which LPS-HFD groups included a low-dose (150 µg/kg) lipopolysaccharide–high-fat diet (LD-LPS-HFD) and a high-dose (200 µg/kg) lipopolysaccharide–high-fat diet (HD-LPS-HFD) group. AC group rats were intraperitoneally injected with 0.9% physiological saline solution and fed ordinary feed; while LPS-HFD groups were intraperitoneally injected with LPS twice a week and had a high-fat diet for 8 weeks. Sarcopenia index (SI), relative grip strength, hematoxylin & eosin staining, Sirius red staining, western blotting, and enzyme-linked immunosorbent assay verified sarcopenia.

Results

SI values decreased in LPS-HFD groups and the differences were more than twice the standard deviation of the AC group. Regard to relative grip strength, only the difference in HD-LPS-HFD group was more than twice the standard deviation of the AC group. Cross-sectional areas and fiber diameters of LPS-HFD groups decreased, but were lower in the HD-LPS-HFD group than the LD-LPS-HFD group. MuRF1, FbX32, and p53 in LPS-HFD groups, and p21, IL-6, and TNF-α in the HD-LPS-HFD group increased, but were higher in the HD-LPS-HFD group than the LD-LPS-HFD group.

Conclusion

Sarcopenia is induced by peritoneal injection of LPS (200 µg/kg) and a high-fat diet for 8 weeks in 10-month SD male rats. This model is suitable to study the prevention and treatment of sarcopenia and its molecular mechanisms.

Sarcopenia

Lipopolysaccharide

High-fat-diet

Atrophy

Fibrosis

Inflammation

Sarcopenia is an age-related, progressive, and systemic disease resulting from a sustained decrease in muscle mass, strength, and physical performance (Cruz-Jentoft et al. 2019). In 2016, sarcopenia was classified under the International Classification of Diseases Code (ICD-10-CM M62.84), which was recognized in Australia in 2019 (Anker et al. 2016; Zanker et al. 2020). Epidemiology has shown that the prevalence of sarcopenia ranges from 0.2–86.5% worldwide (Petermann-Rocha et al. 2022). It is predicted that by 2050, sarcopenia will affect more than 500 million common in older adults (Cruz-Jentoft et al. 2010), muscle mass begins to decline after the age of 40 (Cruz-Jentoft et al. 2019). Thus, sarcopenia may affect the quality of life and health care needs of both middle-aged and older people (Cruz-Jentoft et al. 2019; Petermann-Rocha et al. 2020). It is not only a major health problem but also a significant burden on global healthcare systems.

In addition, the definition and diagnosis of sarcopenia have been constantly updated (Cruz-Jentoft et al. 2010; Cruz-Jentoft et al. 2019; Chen et al. 2020), so it can be seen that sarcopenia has attracted more attention in international community (Yuan et al. 2022; Xiao et al. 2022). To facilitate research, suitable animal models of sarcopenia that are conducive to the study of the pathophysiological basis, molecular mechanism, and potential intervention points are needed (Zhou et al. 2022; Li et al. 2022). With the continuous development of research on sarcopenia, modeling methods have gradually diversified, and literature reports include natural aging, suspension, drug injection, surgery and, knockout gene technology (Palus et al. 2017; Li et al. 2022). However, each of these molding methods has certain limitations. For example, natural aging models require a large amount of time and cost (Palus et al. 2017), the characteristics of sarcopenia exhibited by knockout gene technology may not be observed under normal aging conditions, and knockout can only examine a few specific pathways at a time (Li et al. 2022). In addition, the animal model of hind limb unloading is an animal model of sarcopenia related to resting rather than natural aging of the skeletal muscle, which cannot completely replicate human sarcopenia (Palus et al. 2017). Lipopolysaccharide (LPS), one of the most studied immune-stimulating composition of bacteria, is widely applied in induce immune disorders (Orellana et al. 2006; Liu et al. 2013). Several studies have demonstrated that LPS causes skeletal muscle atrophy and induces sarcopenia (Wan et al. 2017; Liang et al. 2021). Researchers have found that nutrition-related muscle atrophy, such as LPS-induced muscle atrophy, is mainly limited to rapidly contracting glycolytic fibers, and its potential mechanisms are concerned with aberrant protein degradation. Furthermore, multiple studies have used a high-fat diet (HFD) alone to establish a sarcopenia model (Tang 2017; Garcia-Contreras et al. 2018). Long-term intake of high-fat foods can induce insulin resistance (IR) and the expression of pro-inflammatory cytokines, and accelerate the development of sarcopenia (Rasool et al. 2018).

Therefore, this study used an intraperitoneal injection of LPS and high-fat feeding intervention for 8 weeks to accelerate the aging of male Sprague-Dawley (SD) rats aged 10 months to established a new animal model of sarcopenia based on middle-aged male rats. This model lays the foundation for future research on the prevention and treatment of sarcopenia.

2.1 Animals and experimental environments

We purchased 18 9-month-old healthy male SD rats (body weight: 590–725 g) from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Certificate No.: SCXK (Lu) 20190003). All rats were fed at a temperature of 22°C and humidity of 40%-60%, and were subjected to a 12-h dark/light cycle (19:00–7:00). After 1 month of adaptive feeding, the rats were randomly divided into an adult control group (AC group, n = 6), a low-dose LPS-high-fat diet group (LD-LPS-HFD group, n = 6), and a high-dose LPS-high-fat diet group (HD-LPS-HFD group, n = 6). This study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (No. FJTCM IACUC 2022138).

2.2 Animal modeling methods

(1) AC group: intraperitoneal injection of 0.9% physiological saline solution was administered twice a week (Tuesday and Friday) for 8 consecutive weeks, and ordinary feed was provided every day; (2) LPS-HFD groups: 10 mg LPS (L2880; Sigma, USA) was dissolved in 20 ml 0.9% physiological saline solution then the LPS groups were administered intraperitoneal LPS injections of 150 µg/kg (LD-LPS group) (22–24) or 200 µg/kg (HD-LPS group) bi-weekly (Tuesdays and Fridays) for 8 weeks. Each animal was administered 20–30 g of 60% high-fat food daily (D12492; Puning, China).

2.3 Sample preservation

After the gastrocnemius was rinsed with physiological saline solution, part of it was placed at -80°C; part of it was fixed in 4% paraformaldehyde solution, preserved at 4°C for 24–48 h, dehydrated with gradient alcohol, and then embedded to make paraffin sections for histological examination.

2.4 Sarcopenia index (SI) value of target muscle

The gastrocnemius muscle of rats was used as the target muscle for the experiment, and the SI value was obtained as the ratio of the bilateral gastrocnemius mass to the final body weight.

2.5 Grip strength test

A non-invasive grip dynamometer (XR-YLS-13A; XinRuan, China) was used to measure forelimb grip strength of the rats before sampling. The forelimbs of the rats were held onto a metal mesh (23 × 25 cm) and their tails were pulled back at an even speed until their claws came off the mesh, which measured the maximum contractile force of the forelimbs during autonomous motion. The measurements were repeated three times for each rat, and the maximum value was forelimb grip strength. However, to eliminate the effect of body weight on skeletal muscle strength, relative grip strength (maximum grip strength/body weight) was used in our study as an indicator to evaluate the skeletal muscle strength of the rats (Zhu et al. 2020).

2.6 Hematoxylin & eosin (H&E) staining

H&E staining was applied to observe skeletal muscle morphology in rats. After dehydration and embedding in paraffin, tissues were cut into paraffin sections with a thickness of 5 um. Sections were dewaxed, hydrated with xylene and gradient alcohol, and stained using an H&E staining kit (G1003; Solarbio, China). Hematoxylin is an alkaline solution that dyes the nucleus blue; eosin is an acidic dye, giving the cytoplasm a pink color. After dyeing, the cover glass was covered and observed under a light microscope. Image Pro-Plus was applied to analyze the mean cross-sectional area and diameter of gastrocnemius muscle to compare changes in the skeletal muscle morphological structure.

2.7 Sirius red staining

Sirius red staining was used to assess interstitial fibrosis in the rat skeletal muscles; pink collagen fiber and yellow muscle cells are visible after staining. As mentioned above, after dewaxing and hydration of the 5 µm paraffin sections, staining was performed using a Sirius Red dyeing kit (G1470; Solarbio, China). After covering with a cover glass, staining of the detected site was observed under an optical microscope. ImageJ was used to analyze the ratio of collagen-positive area to cell area to calculate the collagen volume fraction, and the content of collagen fiber tissue in gastrocnemius of rats was compared to distinguish the degree of skeletal muscle fibrosis.

2.8 Western blot

The protein levels of MuRF1, Fbx32, p21, and p53 in frozen gastrocnemius tissues were detected. Tissue homogenates were prepared and proteins were quantified using a bicinchoninic acid protein assay. The same contents of protein were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane under wet conditions. After the closure, primary and secondary antibody incubation was completed, the membrane was soaked in chemiluminescent reagent, and the membrane was imitated using a biological image analysis system. Finally, Image Lab software was applied to analyze the gray values of protein bands. Primary antibodies included the following: anti-MuRF1 (sc-398608), anti-Fbx32 (ab168372), anti-p21 (ab109199), anti-p53 (ab183544), and GAPDH (60004-1-Ig; Proteintech, China).

2.9 Enzyme-linked immunosorbent assay (ELISA)

The frozen gastrocnemius tissues were weighed and cut into pieces. Then, 10 ml 0.9% physiological saline solution was added to every 1 mg of rat skeletal muscle tissue for thorough grinding to homogenize the tissue. After the supernatant was extracted using centrifugation (4 ℃, 14,000 rpm, 15 min), the levels of skeletal muscle inflammatory factors were determined according to the ELISA kit requirements. Two kits were used in this study: pro-inflammatory interleukin-6 (IL-6; EK0412; BOSTER, China) and tumor necrosis factor-alpha (TNF-α; EK0526; BOSTER, China).

2.10 Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics 23.0 (Armonk, NY, USA). Data was expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was applied to compare groups. The least significant difference test was used for pairwise comparisons with the variance homogeneous, while the Games-Howell test was used with the variance unhomogeneous. Differences were considered statistically significant when the P-value was < 0.05.

3.1 SI values of gastrocnemius muscle

In comparison with the AC group, the SI values of the LD-LPS-HFD (P < 0.01) and HD-LPS-HFD groups (P < 0.001) were significantly reduced, and the difference was more than twice the standard deviation of the AC group; while HD-LPS-HFD group had more declined (P < 0.05) (Fig. 1A).

3.2 Relative grip strength

After comparing the final relative grip strength of the three groups, it was found that both e of the LD-LPS-HFD (P < 0.01) and HD-LPS-HFD groups (P < 0.001) were significantly lower than that in the AC group, and the decrease of HD-LPS-HFD group was greater(P < 0.05); however, only the discrepancy between the HD-LPS-HFD and AC groups was greater than twice the standard deviation of the AC group (Fig. 1B).

3.3 H&E staining of gastrocnemius muscle

As shown in Fig. 2A, the AC group showed normal skeletal muscle histological features, with an orderly arrangement of muscle fibers and no myocyte edema, polygons in cross sections, regular shapes, uniform sizes, multiple nuclei located at the cell membrane, and a clear cell membrane boundary. In the LD-LPS-HFD group, the arrangement of muscle fibers was loose, the muscle cells were round, and the individual nuclei were centralized; however, the membrane boundaries were still clear. The HD-LPS-HFD group showed a loose arrangement of muscle fibers, a reduced number of muscle fibers, myocyte edema, centralization of most nuclei, and blurred membrane boundaries. By comparing the cross-sectional areas and fiber diameters of three groups, the results indicated that those of the LD-LPS-HFD group (P < 0.001 or P < 0.01) and HD-LPS-HFD group (P < 0.001) were smaller than those of the AC group. And the largest reduction was observed in HD-LPS-HFD group (P < 0.001) (Fig. 2B, 2C).

3.4 Fibrosis of gastrocnemius muscle

As shown in Fig. 3A, there was evident infiltration of skeletal muscle interstitial fibers in LPS-HFD groups. In comparion with the AC group, the skeletal muscle collagen volume fraction of the LD-LPS-HFD group (P < 0.001) and HD-LPS-HFD group (P < 0.01) obviously increased. And the the HD-LPS-HFD group had more reduction (P < 0.05) (Fig. 3B).

3.5 Expression of muscle fiber atrophy factors in the gastrocnemius muscle of rats

In comparison with the AC group, both MuRF1 and FbX32 were remarkably increased in the LD-LPS-HFD (P < 0.01) and HD-LPS-HFD (P < 0.01 or P < 0.001) groups; the largest enhancement was found in the HD-LPS-HFD group (P < 0.05 or P < 0.001) ( Fig. 4A, 4C, 4D).

3.6 Expression of aging markers in the gastrocnemius muscle of rats

The contents of p21 and p53 in the LD-LPS-HFD group were more than in the AC group, but only the difference in p53 was significant (P < 0.01). Conversely, p21 and p53 in the HD-LPS-HFD group were obviously more than in the AC and LD-LPS-HFD groups (P < 0.001) (Fig. 4B, 4E, 4F).

3.7 Expression of gastrocnemius inflammatory factors in rats

The expression levels of IL-6 and TNF-α in the LD-LPS-HFD group were not different from those in the AC group (P > 0.05). However, they were significantly increased in HD-LPS-HFD group (P < 0.01) and greater than the LD-LPS-HFD group (P < 0.05)(Fig. 5A, 5B).

In this study, we investigated the effects of LPS-HFD on the development of sarcopenia in rats. The major findings were that after 8 weeks of intervention with LPS-HFD, 12-month-old rats (sarcopenia model rats) not only exhibited a sarcopenia phenotype consistent with decreased muscle strength and decreased muscle mass, but also exhibited a range of features of aging.

In the light of the European Working Group on Sarcopenia in Older People 2 (EWGSOP2), decreased muscle mass, strength, and physical performance could be considered as criteria for the diagnosis of sarcopenia (Cruz-Jentoft et al. 2019). Previous studies have used the SI and relative grip strength to evaluate rat sarcopenia models (Edström and Ulfhake 2005; Zhou et al. 2018). When the SI value of model rats decrease obviously compared with that of control, and the difference is greater than twice the standard deviation of the control group, sarcopenia was determined, indicating successful modeling (Edström and Ulfhake 2005; Zhu 2020). Our data showed that SI values and relative grip strength of two LPS-HFD groups were significantly reduced, and the differences between the SI value of LPS-HFD and the relative grip strength of HD-LPS-HFD were significantly greater than twice the standard deviation of the AC group, indicating that both models were successfully constructed, but only the high-dose group showed changes in muscle strength decline due to sarcopenia. Ko et al. (Ko and Ko 2021) found that aging resulted in muscle fiber atrophy and reduced the SI value of the gastrocnemius muscle. Jin et al. (Jin et al. 2023) indicated that the maximum grip strength of sarcopenia model rats was observably less than control group. Therefore, our model of sarcopenia induced by LPS-HFD was consistent with the presentation of sarcopenia, with the HD-LPS-HFD group having a better modeling effect that was more consistent with sarcopenia.

Skeletal muscle fiber atrophy is the main pathological feature of sarcopenia, and the cross-sectional area and diameter of the fibers are the most intuitive manifestations of skeletal muscle atrophy (Shang et al. 2020). In this study, we found that the muscle fiber cross-sectional areas and diameters were smaller in two LPS-HFD groups, while those of the HD-LPS-HFD group were less than those of the LD-LPS-HFD group. We found that the E3 ligases associated with muscular atrophy, such as MuRF1 and FbX32 (also known as Atrogin-1, MAFbx), were significantly elevated in the skeletal muscle of the two LPS-HFD groups, and increased with dose, suggesting that protein degradation and increased muscle atrophy were closely related.

FbX32 and MuRF1 are skeletal muscle-specific E3 ligases that inhibit skeletal muscle protein synthesis and are significantly upregulated in a variety of skeletal muscle models (Foletta et al. 2011; Zhu et al. 2013). Study found that in the skeletal muscle of SAMP8 mice, markers related to protein synthesis (Akt and p70S6K) decreased, whereas markers related to protein degradation (FoxO3, MuRF-1 and MAFbx) significantly increased, indicating that protein imbalance in the aging process makes decomposition greater than synthesis, inducing the occurrence of sarcopenia (Liu et al. 2020). Simultaneously, Atrogin-1 and MuRF1 showed similar changes in various aging tissues, with significant increases (Ziaaldini et al. 2015; Kou et al. 2017).

However, the increase in skeletal muscle interstitial fibrous tissue is major histopathological change in sarcopenia and is an important reason for the decline in skeletal muscle weakness and movement in elderly individuals (Lyu et al. 2019; Shang et al. 2020). Our study found that the collagen volume fraction of the gastrocnemius in the two LPS-HFD groups was increased markedly, and more interstitial fiber tissue deposition occurred in the HD-LPS-HFD group. Previous study has shown that an increase in type I collagen restricts the contraction of skeletal muscles and weakens the ability of skeletal muscles to adapt to changes in external load, thereby reducing exercise capacity (Mahdy 2019). In aging mice, the increase in type I collagen is particularly significant (Lyu et al. 2019; Shang et al. 2020). In addition, the deposition of fiber tissue may interfere with the interaction between satellite cells and surrounding cells, affecting the regeneration of muscle tissue, increasing susceptibility to skeletal muscle reinjury, and further aggravating skeletal muscle atrophy (Stearns-Reider et al. 2017).

Moreover, replicative senescence and stress-induced premature senescence (SIPS) are the types of cellular senescence (Liang et al. 2022). SIPS is induced by multiple stress factors such as DNA damage, oxidative stress, and inflammation (Xie et al. 2021; Calcinotto et al. 2019), which are mainly triggered by the regulation of the p53-p21 and p16INK4a-pRB signaling pathways (Xie et al. 2021). Among them, p53-p21, with p53 as the central link, is the most critical pathway that could lead to age-related phenotypes such as muscle atrophy (Qian et al. 2013; Wu et al. 2020). Yoshida et al. found that p53 and p21 were enhanced in muscle tissues of mice with sarcopenia, which is consistent with the aging characteristics of mice (Yoshida et al. 2019; Huang et al. 2021). In addition to the sarcopenia model, Calcinotto et al. demonstrated that aging biomarkers, such as p21 and p53, were significantly elevated in senescent cells (Calcinotto et al. 2019). These results confirm that aging and sarcopenia can lead to high p53 and p21 expression, which is in accordance with our study. Our study found that p53 and p21 were highly expressed in the gastrocnemius muscle of LPS-HFD group, and the HD-LPS-HFD group showed higher expression and more severe aging.

Numerous investigations indicate that chronic inflammation is a favorable environment for the evolution of sarcopenia (Dalle et al. 2017; Tournadre et al. 2019). The major factors involved in inflammation include TNF-α, IL-6, and chemokines, which promote inflammatory cell infiltration and muscle deterioration through NF-κB (Zhang et al. 2022). Among them, IL-6 and TNF-α are non-specific markers of frailty and aging; are negatively correlated with muscle strength, muscle mass, and function (Rodriguez-Mañas et al. 2020), which are closely related to the pathogenesis of sarcopenia (Liang et al. 2022). Rong et al. found that IL-6 could increase muscle catabolism, and was positively correlated with sarcopenia (Rong et al. 2018). In addition, some scholars have observed that the level of TNF-α was remarkably increased in older patients with sarcopenia (Li et al. 2019), and the reduction of TNF-α in the whole body could prevent sarcopenia (Wang et al. 2018). This was consistent with our results, which showed that both IL-6 and TNF-α levels were increased in HD-LPS-HFD group, but not in LD-LPS-HFD group. This further indicates that the HD-LPS-HFD group showed more evident aging.

In summary, this study developed a new modeling method of sarcopenia by intraperitoneal injection of LPS and a high-fat diet administered to 10-month-old male rats for 8 weeks. The SI value, relative grip strength, tissue morphological changes, fibrosis, inflammation and aging markers were used to evaluate the models.

Strengths and limitations

This study is innovative in two ways: (1) existing studies have not injected 200 µg/kg LPS into 10-month-old rats, and (2) the combination of LPS and a high-fat diet to induce sarcopenia has not been studied. Nevertheless, not using naturally aging rats as a control group is a limitation of this study.

The present study indicates that sarcopenia can be induced by an intraperitoneal injection of 200 µg/kg LPS and a high-fat diet in 10-month-old male SD rats for 8 weeks. The model rats showed sarcopenia phenotypes including muscle strength and muscle mass reduction, cell atrophy, cell nucleus centralization, interstitial fiber infiltration, increased inflammation, and high expression of aging markers. In terms of study limitations, if time and financial resources are sufficient, future studies could use an additional group of naturally aging rats as controls to better compare the differences in the model.

SD, Sprague–Dawley; AC, Adult Control; LPS, lipopolysaccharide; HFD, high-fat diet; LD, low -dose; HD, high-dose; SI, Sarcopenia index; H&E, Hematoxylin & eosin; PVDF, polyvinylidene fluoride; ELISA, Enzyme-linked immunosorbent assay; IL-6, pro-inflammatory interleukin-6; TNF-α, tumor necrosis factor-alpha; ANOVA, One-way analysis of variance; EWGSOP2, European Working Group on Sarcopenia in Older People 2.

Funding

This work was supported by National Natural Science Foundation of China (grant number 81904317); Natural Science Foundation of Fujian province (grant number 2023J01759); and High-level talent start-up project of university (grant number XRCZX2022036).

Declaration of competing interest

All authors claim that there are no conflicts of interest.

Author contribution

GYF, CSQ, WSZ and LJP conceived and designed the project. GYF, LM, WY and CBR performed experiments. GYF and LM analyzed data and prepared figure. GYF drafted the manuscript. CSQ, WSZ and LJP supervise the entire experimental process. LJP provide resources. CSQ, WSZ and LJP edited and revised the manuscript. All authors contributed to the article approved the final version of the manuscript. GYF and LM contributed equally to this work.

Data availability

The data used to support the findings of this study are available on request from the corresponding author.

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Establishment and Evaluation of a Rat Model of lipopolysaccharide-high-fat diet Induced Sarcopenia

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Abstract

Objective

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1 Animals and experimental environments

2.2 Animal modeling methods

2.3 Sample preservation

2.4 Sarcopenia index (SI) value of target muscle

2.5 Grip strength test

2.6 Hematoxylin & eosin (H&E) staining

2.7 Sirius red staining

2.8 Western blot

2.9 Enzyme-linked immunosorbent assay (ELISA)

2.10 Statistical analysis

3. Results

3.1 SI values of gastrocnemius muscle

3.2 Relative grip strength

3.3 H&E staining of gastrocnemius muscle

3.4 Fibrosis of gastrocnemius muscle

3.5 Expression of muscle fiber atrophy factors in the gastrocnemius muscle of rats

3.6 Expression of aging markers in the gastrocnemius muscle of rats

3.7 Expression of gastrocnemius inflammatory factors in rats

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

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