Sarcopenia, a loss of muscle mass accompanying osteoporosis, leads to falls and fall-related injuries. Baicalein, as a phytochemical agent, has an antioxidative and anti-inflammatory effect in muscle. In this study, sixty-one female Sprague Dawley rats were divided into five groups: four groups were ovariectomized (OVX) and one control group was nonovariectomized (NON-OVX). Eight weeks after ovariectomy, three disparate concentrations (1 mg/kg body weight (BW), 10 mg/kg BW, and 100 mg/kg BW) of baicalein were applied subcutaneously daily in three OVX groups. Mm. soleus, gastrocnemius, and longissimus were extracted; their diameter, area, relation to body, and muscle weights as well as number of capillaries per fibre were recorded. In Mm. soleus and gastrocnemius, the baicalein effect (increasing number of capillaries per fibre) was proportional to the dose applied. The fibre diameters and area under baicalein treatment were significantly greater compared to OVX and NON-OVX groups. In M. longissimus, we observed a shift to type IIa fibres. Serum creatine kinase levels were significantly lower in highest baicalein concentration group. We conclude that baicalein can stimulate angiogenesis, though not fibre type-specific, in skeletal muscle and reduce the estrogen-related loss of fibre diameter and area in the skeletal muscle in rats. Therefore, a protective effect of baicalein on muscle cells can be assumed.
Osteoporosis is one of the most common diseases in humans. Recent data assume lifelong prevalence of about 11.9% of osteoporosis in Germany [
Sarcopenia, a loss of skeletal muscle mass, emerges during the development of osteoporosis [
Former studies suggested that a low grade inflammation contributes to sarcopenia [
Therefore, therapeutic possibilities reside in pharmacological intervention which target impaired regenerative capacity, elevated reactive oxygen species production, and inflammation observed in sarcopenic muscle [
Baicalein, as a phytochemical agent, is extracted from the plant Scutellaria baicalensis Georgi. It acts as a lipoxygenase (especially cyclooxygenase (COX-I) inhibitor) and is also a potent inhibitor to 12-lipoxygenase (12-LOX) and 15-LOX and, therefore, has an antioxidative effect [
For the study, 61 female, 3-month-old Sprague Dawley rats (Winkelmann Company) were kept in cages at 20°C and at a relative humidity of 55% in Makrolon IV®. After 1 week of acclimatization, the experiments were conducted in accordance with the ethical standards of animal care and approved by the local district government (application number: G14/1530).
At 13 weeks of age, the rats underwent bilateral ovariectomy (OVX) or were left intact, as previously described [
The animals were sacrificed after the baicalein treatment. M. gastrocnemius, M. soleus, and M. longissimus were removed, and M. gastrocnemius and M. soleus were weighed. M. soleus was cut 4-5 cm before the foreleg. Afterwards, M. gastrocnemius and M. soleus were sectioned transversely in half across the whole muscle to make cryotome-cutting easier.
The muscles were frozen in liquid nitrogen and stored at −80°C. Subsequently, 12
To identify the distribution and changes in types I and II fibres, ATPase staining was carried out on muscle sections, as described by Horák [
PAS and ATPase staining of M. longissimus ((a) and (c)) and M. gastrocnemius (b). The PAS staining of M. longissimus shows capillaries marked by arrows (a). In ATPase staining, type I fibres are enclosed in red, IIa fibres in black, and IIb fibres in orange in M. gastrocnemius, whereas example fibres are marked by black arrows (b); in M. longissimus, type I fibres are marked by a red arrow (surrounded by a red shape), type IIa fibres with a black arrow (surrounded by a black shape), and IIb fibres with a yellow arrow (surrounded by a yellow shape) (c) (100x magnification).
For capillary counting, two sections of each muscle in PAS staining were chosen and, in a 0.25 mm2 square, every capillary and muscle fibre were counted, which taken together delivered the ratio of capillary/muscle fibre.
For fibre measurements, in three different image sections of each muscle in ATPase staining, 30 type I, 30 type IIa, and 30 type IIb fibres were edged and area and diameter were determined by the program NIS-Elements AR 4.0 (Nikon Instruments Europe, Amsterdam, Netherlands). After that, in 1 mm2 fibre, types were counted and their relation calculated.
After the rats were sacrificed, blood samples of 0.5 mL were collected and analysed with the creatine kinase assay using a c16000 analyser (Abbott, Wiesbaden, Germany) for quantitative measurement of serum values. Calcium was measured with the same analyser at 660 nm using Arsenazo III dye and magnesium at 572 nm after the calcium was complexed and eliminated with a chelating agent so as to not interfere with the measurements.
Statistical analyses were conducted using GraphPad Prism (version 5.04, GraphPad Software, Inc., San Diego, CA). For detecting differences between the different groups, one-way ANOVA (
To test the effects of ovariectomy and different concentrations of baicalein on body weight, the rats were weighed on the day of receipt (Figure
Body weight in the beginning (a) and end (b) of trial, weight of uteri (c), M. gastrocnemius (d), and M. soleus (e). The body weight of rats was monitored on the days of delivery ((a),
There was no significant difference between groups in body weight on the day of receipt. However, a significant difference in postmortem weight was noted between the NON-OVX and OVX groups. The NON-OVX group was significantly lighter than the OVX group.
The postmortem weights of M. gastrocnemius (Figure
To test the effects of ovariectomy on the uterus, the uteri were extracted and weighed (Figure
In a previous study, aging was correlated with a decrease in the number of capillaries per muscle fibre [
Number of capillaries per single muscle fibre in M. soleus, M. gastrocnemius, and M. longissimus. The numbers of capillaries per muscle fibre in M. soleus ((a),
Although we could not see differences between NON-OVX and OVX animals, rats treated with baicalein showed significantly higher numbers of capillaries per muscle fibre when compared with the untreated groups. On average, the rate of capillarization could be increased between 20% and 40% when rats were treated with baicalein. Moreover, the group treated with the highest concentration of baicalein (C3) showed significantly more capillaries per fibre when compared with the group treated with the lowest concentration (C1).
After ATPase staining, muscle fibres were analysed for their diameter and area. In total, 90 fibres of each type for each sample were assessed, and 90% of all cells were type I; therefore, no further differentiation was performed.
To assess characteristics of sarcopenia, we determined the diameters and areas of the muscle fibres.
For M. soleus, fibre diameter was determined and analysed in relation to body weight. Although the diameter in NON-OVX rats was on average 63
M. soleus fibre diameter and area and their relation to body weight. The diameter of single fibres was increased in each baicalein C2 and baicalein C3 group versus the OVX group ((a),
When relating muscle fibre diameter and area to muscle weight, no significant differences, but similar tendencies to the relation to body weight, were seen. This is perhaps because of relatively low muscle weight, which could lead to measurement inaccuracy (Figures
In M. gastrocnemius, the distribution of different fibre types is very heterogeneous. We assessed the three fibre types—I, IIa, and IIb—for their diameter and area. Type I fibres showed an increased diameter of approximately 20% when treated with baicalein compared with the NON-OVX group (Figure
Diameter and area of M. gastrocnemius fibre types I and IIa and their relation to muscle and body weights. The diameter of single type I fibres was significantly greater in the baicalein C2 and baicalein C3 groups compared with the NON-OVX group ((a),
Regarding the size of the area of different fibre types, in type I (Figure
Thereafter, we calculated the diameter to body weight ratio and found no significant differences in types I (Figure
Moreover, we calculated the relation of fibre area to body weight. In type IIa fibres, we found a slight increase in M. gastrocnemius area in group C2 when compared with OVX rats; however, other groups did not show significant differences (Figure
When relating area to muscle weight, we found significant differences in type I fibres, where NON-OVX, OVX, and C1 groups did not show significant differences (Figure
Comparable with M. gastrocnemius, the three fibre types could also be differentiated for M. longissimus. When observing the diameter in M. longissimus type I fibres, we did not detect significant differences between NON-OVX and OVX rats; however, high doses of baicalein (C2) could raise the diameter by approximately 13% (Figure
Diameter and area of M. longissimus fibre types I and IIa and their relation to muscle weight and body weights. A significantly greater fibre type I size was noted in the C2 group than in the NON-OVX, the OVX group, and the C1 group ((a),
Similar effects were observed in type IIa fibres, where baicalein C2 could increase the diameter to 86.7
We measured the area of muscle fibres types I and IIa and, analogous to the diameter, similar values were obtained for NON-OVX and OVX animals (Figures
Unlike the analysis of M. soleus and gastrocnemius, we related the fibre diameter and area in M. longissimus to animal body weight because single muscle weight was not detected. Type I fibre diameter was significantly decreased after ovariectomy (Figure
When relating the areas of types I and IIa fibres to body weight, a similar decrease could be observed in OVX rats when compared with the NON-OVX control group (Figures
Finally, the proportions of different fibre types (type I, IIa, and IIb) were assessed in M. longissimus (Figure
Rate of M. longissimus fibre types I, IIa, and IIb. In M. longissimus, the distribution of fibre type I was not significantly different among the groups ((a),
In M. soleus and M. gastrocnemius, fibre type distribution was not determined.
Creatine kinase (U/L), calcium (Ca2+; mmol/L), and magnesium (Mg2+; mmol/L) levels were measured in the groups. Creatine kinase levels were significantly lower in the C3 group than in the OVX group (Figure
Serum creatine kinase, calcium, and magnesium levels. Compared with the OVX group, the C3 group had a significantly lower creatine kinase level ((a),
Sprague Dawley rats are a valuable model for the induction of osteoporosis after hormonal depletion via ovariectomy [
Independent of baicalein treatment, body weight of the rats after ovariectomy was significantly greater for the OVX (control) group than for the NON-OVX group. Application of baicalein could partially rescue the effect of estrogen deficiency in muscle. Interestingly, in M. gastrocnemius, an increasing dose of baicalein led to slightly reduced muscle weight, supporting the hypothesis of estrogen deficiency-induced muscle degeneration. However, the reduced muscle weight of M. gastrocnemius could also be attributed to the baicalein-induced inhibition of the nonphysiological degeneration of muscle, a finding reported in literature describing the anti-inflammatory and antifibrotic effects of baicalein [
As reported previously, estrogen has an angioproliferative effect on skeletal muscle [
The expected loss of capillaries under estrogen deficiency could not be observed when treated with baicalein. The hypothesis that baicalein, applied subcutaneously, stimulates angiogenesis in skeletal muscle despite estrogen deficiency was proven. Nonetheless, there were no significant differences in capillary to muscle fibre ratio after OVX, which might be due to insufficient trial duration, which was too short to reveal the decapillarization in sarcopenic muscle, as seen in other rat models [
We purported that decreased muscle diameter due to estrogen loss, as described in rats [
In all three fibre types (I, IIa, and IIb) in M. gastrocnemius we saw a tendency of all baicalein-treated groups to have increased fibre diameters. Interestingly, the group treated with baicalein C2 concentration showed the greatest fibre diameters.
When analysed in detail, the diameter to body weight ratio showed that types I, IIa, and IIb fibres in almost all baicalein groups were slightly increased. We also found a tendency for greater diameter per body weight values in the NON-OVX group when compared with the OVX group. Overall, baicalein appears to induce hypertrophy in the estrogen-deficient skeletal muscle.
In M. longissimus, the development of sarcopenia after ovariectomy, resulting in estrogen deficiency, which leads to reduced muscle fibre diameter is supported. The baicalein C2 group showed the highest fibre diameters.
Altogether, the expected atrophy of skeletal muscle fibres, indicated by the decline in fibre diameter [
In M. longissimus, the increase in types I and IIa fibre areas suggests that baicalein can counteract the estrogen-dependent atrophy. In M. gastrocnemius, the increase of type I fibre area suggests the same, especially at the C2 concentration. Nonetheless, this gain in area and diameter can also be attributed to the storage of fat and water [
In M. longissimus, significantly increased proportions of fast IIa but not type I fibres were observed in the baicalein C2 group. This finding disagrees with the decline in fast fibres, the described “fast-to-slow fibre type shift,” which is characteristic for muscle atrophy and wasting [
In conclusion, these results suggest a positive, though not fibre type-specific, effect of baicalein on estrogen-deficient muscles in rats. Our hypothesis that baicalein promotes angiogenesis in muscle could be confirmed. Whereas in M. gastrocnemius and M. soleus the number of capillaries in muscle was proportional to the dose of baicalein, in M. longissimus, the medium baicalein concentration (10 mg/kg BW) showed the greatest effect. The results of muscle diameter and area also showed a positive baicalein effect in comparison with the control groups. Moreover, regarding fibre ratios, a gain in IIa fibres was seen in M. longissimus, which contradicts the “fast-to-slow fibre type shift.”
As observed in human studies, there was no difference between sarcopenic and control groups regarding calcium and magnesium levels [
Serum concentration of creatine kinase, a clinical marker of muscle state [
Investigating molecular mechanisms of baicalein treatment was not within the scope of this descriptive study, but Bhattacharya et al. showed that skeletal muscle atrophy (during aging and neurodegenerative diseases) can be diminished with inhibition of 12-LOX and 15-LOX, while the activation of this pathway led to increased NADPH oxidase activity, protein ubiquitination, and proteolytic degradation. They also demonstrated that baicalein reduces denervation-induced muscle atrophy in wild type but not in 12-LOX and 15-LOX knockout mice. Therefore, baicalein chiefly operates through this pathway and is not arbitrated through the reduction of inflammatory response. Possibly the main effect of baicalein in this study could be observed because of the above-mentioned 12-LOX and 15-LOX pathway inhibiting NADPH oxidase activity and protein ubiquitination as well as “ubiquitin-proteasome-mediated proteolytic degradation” [
The aim of this study was to elucidate whether baicalein, in different doses, applied subcutaneously, could neutralize the estrogen-induced loss of muscle and atrophy in the skeletal muscle of rats. We found that baicalein has a positive effect on estrogen-dependent atrophy of skeletal muscle.
We injected baicalein subcutaneously, which resulted in necrotic lesions at the injection sites. This could be seen in baicalein and not in DMSO-treated rats. This inflammatory effect has not been reported so far but did not affect overall health, as was seen by the analyses of body weight, food intake, and general conditions (clean coat, absence of porphyrin around eyes, etc.). The inflammatory processes were not part of this study and we could not confirm an anti-inflammatory effect after s.c. injection of baicalein as it was shown after experimental traumatic brain injury [
To emphasize differences between baicalein and OVX groups, trial duration could be elongated and more groups could provide better conditions to find the ideal dose and route of baicalein administration. Moreover, a healthy NON-OVX group should be included. As we only investigated skeletal muscle, we cannot exclude systemic effects. No fever or severe infections occurred throughout the experiments. However, the development of the necrotic lesions at the injection sites should be taken into consideration. Smooth muscles and cardiomyocytes as well as infection parameters, such as CRP, IL-6, and procalcitonin, could be further examined to rule out effects on these tissues [
To exclude the fact that gains in area and diameter are attributed to fat and water retention, biochemical analyses of muscle fibres would be useful. Additionally, there could be a possible effect of osteotomy on muscle tissue, so these experiments could be repeated without this operative procedure; however, this effect would exist in all rats.
Different models of muscle inflammation could also be used to further investigate the anti-inflammatory effect of baicalein. In addition, the bone of rats and its biomechanical and histomorphometric composition could also be evaluated, as recently done by Li et al. [
Detection of the molecular mechanism underlying the effects of baicalein in rats was not part of this descriptive study. Therefore, further examinations including
Baicalein increased capillaries per muscle fibre but not total muscle weight. This discrepancy could be explained by baicalein-induced inhibition of the nonphysiological degeneration of muscle due to the anti-inflammatory, antifibrotic [
Protein kinase b
Adenosine triphosphate
Body weight
Cyclooxygenase
C-reactive protein
Dimethyl sulfoxide
Extracellular signal-regulated kinase
Hypoxia-inducible factor 1-alpha
Insulin-like growth factor 1
Interleukin
Interleukin-6
Intraperitoneal
Lipoxygenase
Mechanistic target of rapamycin
Nicotinamide adenine dinucleotide phosphate
Nuclear factor kappa-light-chain-enhancer of activated B cells
Nonovariectomy
Ovariectomy
Periodic acid-Schiff
Standard error of the mean
Tumor necrosis factor-alpha 8
Vascular endothelial growth factor.
M. Wicke is deceased.
The authors declare that they have no competing interests.
The authors’ gratitude goes to Elsbeth Bonhoff Stiftung for the financial support of the present study (Grant N114). The authors are also grateful to R. Wigger, R. Castro-Machguth, and A. Witt for their technical support.