Chinese Herbal Medicine, Guilu Erxian Glue, as Alternative Medicine for Adverse Side Effects of Chemotherapy in Doxorubicin-Treated Cell and Mouse Models

Doxorubicin (DOX), a chemotherapeutic drug, often causes many adverse side effects in patients with cancer, such as weight loss, motor disability, blood circulation defects, myelosuppression, myocardial injury, joint degeneration, and bone loss. The Chinese herbal medicine Guilu Erxian Glue (GEG) has been used in the prevention and treatment of osteoarthritis and osteoporosis for hundreds of years, with considerably fewer side effects. We expected that GEG could serve as a protective and beneficial alternative treatment for DOX-induced adverse side effects. In this study, we evaluated whether GEG can alleviate DOX-induced weight loss, motor disability, abnormal blood circulation, myelosuppression, myocardial injury, joint degeneration, and bone loss by using chemotherapy models of synoviocyte cell line HIG-82 and mice. Moreover, we examined the antioxidant capacity of GEG by using DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical scavenging. Our results revealed that GEG treatment can significantly enhance DPPH free-radical scavenging and reduce DOX-induced cytotoxicity in synoviocyte HIG-82 cells. In addition, GEG treatment for 2 weeks can significantly relieve weight loss, enhance exhaustive exercise capacity, improve blood circulation, alleviate myocardial oxidative stress and inflammation, and strengthen the tibias of DOX-treated mice. Thus, we suggest that GEG treatment can be a protective and alternative therapy for alleviating chemotherapy-related side effects such as weight loss, motor disability, blood circulation defects, and bone loss.

provided cardioprotection in DOX-treated mice through the suppression of oxidative stress, inflammation, and apoptosis [21]. Clinical evidence suggests that DOX induces severe osteoporosis and osteoarthritis in addition to myocardial injury in patients with cancer undergoing chemotherapy [22][23][24][25][26][27]. For example, children receiving DOX chemotherapy might experience long-term bone damage, leading to reduced adult height and increased fracture risk [22]. Premenopausal patients with breast cancer receiving DOX chemotherapy exhibited significantly low bone density and bone loss [23]. In laboratory experiments, DOX chemotherapy caused a 60% reduction in bone formation in normal rats [24,25] and a significant reduction in trabecular bone volume and cortical bone thickness in rabbits [26]. At the cellular level, DOX treatment could inhibit osteoblast cell differentiation in mice [27]. Osteoarthritis often presents risks of falls and disability, whereas osteoporosis often leads to high risks of fractures and subsequent complications in older people and patients with cancer undergoing chemotherapy [28][29][30]. Identifying a potential reliever that can protect from the adverse side effects of chemotherapy without reducing the effectiveness of chemotherapy for patients with cancer receiving DOX treatment is urgent.
Guilu Erxian Glue (GEG) is a typical Chinese herbal medicine used in Taiwan that contains four major components, Cornu Cervi, Testudinis Plastrum, Ginseng Radix, and Lycii Fructus. GEG has been widely applied in the treatment and prevention of osteoporosis and osteoporosis for hundreds of years with remarkably few side effects [31]. In addition, GEG treatment has long-term beneficial effects on aging, perimenopausal syndrome, and degenerative joint disease [32]. In vitro, GEG treatment can stimulate the secretion of IGF-1 in osteoblasts and attenuate bone osteoclast reabsorption [33]. In vivo, GEG treatment can inhibit the formation of osteoclasts and bone pits in rats and reduce articular pain and increase muscle strength in elderly men with knee osteoarthritis [34]. Notably, GEG treatment can prevent and treat myelosuppression following cancer chemotherapy [35]. However, whether GEG can alleviate DOXinduced cardiotoxicity, muscle weakness, osteoarthritis, and osteoporosis has not been fully elucidated.
In this study, we mainly investigated the alleviating effects of GEG treatments in DOX-induced cytotoxicity in synoviocyte cell line HIG-82 and on weight loss, motor disability, blood circulation defects, myelosuppression, myocardial injury, and bone loss in mice subjected to chemotherapy. Our results may provide evidence to suggest that GEG treatment is a protective and beneficial alternative therapy for DOX-induced adverse side effects.

Materials and Methods
2.1. Preparation of GEG. GEG (supplied by Sun-Ten Pharmaceutical Company, New Taipei City, Taiwan) contains four major components, Testudinis Plastrum, Cornu Cervi, Lycii Fructus, and Ginseng Radix, in the ratios of 4 : 2: 2 : 1. All chemical compounds used in this analysis were dissolved in distilled water (H 2 O)/methanol (MeOH). Chromatographic fingerprint analysis was conducted using a 3D high-performance liquid chromatography (HPLC) that mainly followed the methods of our previous study [36].

DPPH Assay of GEG Treatment.
We assessed the antioxidant activities of GEG through a DPPH (1,1-diphenyl-2picrylhydrazyl) assay. As described in the previous study [37], GEG extract (1-20 mg/mL) diluted in distilled water was mixed with 100 μL of 1.5 mM/mL DPPH (D9132, Sigma-Aldrich Co., St. Louis, MO, USA) in methanol in a 96-well plate. After the samples were left to stand for 30 min at room temperature, their absorbance was recorded. e color changes were recorded spectrophotometrically at 517 nm by using a Microplate Spectrophotometer (µQuant, Biotek Intruments, Inc., VT, USA). Appropriate blanks (methanol) and standards (L-Ascorbic acid in water, L-AA; A5960, Sigma-Aldrich) were recorded simultaneously. Each assay was performed in triplicate. e inhibitory activity (%) of DPPH scavenging was calculated using the following expression. DPPH scavenging (%) � 100 × [(absorbance of sample + DPPH) − (absorbance of sample blank)]/[(absorbance of DPPH) − (absorbance of methanol)]. Concentrations of GEG that cause 50% scavenging (IC 50 ) were calculated from the graph in which the scavenging activity was plotted against the corresponding GEG concentration.

Animal Preparation.
A total of 32 five-month-old male ICR (Institute of Cancer Research) mice were purchased from the BioLASCO Breeding Center (AAALAC International awarded, Yi-Lan, Taiwan) and used in this study. Animal experiments were permitted and supervised by the Institutional Animal Care and Use Committee of National Taiwan Normal University (Protocol number: NTNU/Animal Use/No. 109006/Mar. 10, 2020). All experiment protocols were executed in accordance with the international guidelines for the Care and Use of Laboratory Animals. e methods of animal preparation and grouping used in this study mainly followed the methods of our previous study [21]. ICR mice were randomly divided into four groups: sham (negative control group), GEG (positive control group), DOX (DOX treatment group), and DOX + GEG (DOX + GEG treatment group). Mice in the sham group were fed a vehicle (dimethyl sulfoxide) through their drinking water twice daily for 14 days. In the GEG group without DOX treatment, mice were fed GEG extract (30 mg/ mL, pH close to 7.0) through their drinking water twice daily for 14 days. In the DOX group, before DOX treatment, mice were fed a vehicle (dimethyl sulfoxide) through their drinking water twice daily for 14 days and then treated with two intraperitoneal injections of DOX (10 mg/kg body weight). In the DOX + GEG group, before DOX treatment, mice were fed GEG extract (30 mg/mL, pH close to 7.0) through their drinking water twice daily for 14 days and then treated with two intraperitoneal injections of DOX (10 mg/ kg body weight). Our animal experiments were considered to be in accordance with the 3R principles (replace, reduce, and refine) for optimizing the experimental design.

Exhaustive Swimming Experiment Design.
We assessed the exhaustive exercise capacity by conducting the exhaustive swimming experiment in all mice. Sham, GEG-, DOX-, and DOX + GEG-treated mice were subjected to a weight-loaded exhaustive swimming procedure in a swimming tank (50 × 50 × 50 cm 3 ) with 30 cm-deep water maintained at 25 ± 3°C for 1 h. e methods of exhaustive swimming experiment used in this study mainly followed the methods of our previous study [36].

Subcutaneous Microcirculation Measurement.
We assessed the blood circulation function in all mice through subcutaneous microcirculation measurement. A laser Doppler imager (Moor Instruments, Axminister, UK) was used to scan the regional dermal microvascular blood flow of mice in the sham, GEG, DOX, and DOX + GEG treatment groups individually. e methods of subcutaneous microcirculation measurement used in this study mainly followed the methods of our previous study [39]. We selected and averaged the subcutaneous microcirculation measurements for each mouse obtained from at least three stable consecutive laser Doppler images.

Peripheral Blood Smear Analysis.
We assessed the hemogram of all mice by using peripheral blood smear. Blood smear analysis is an integral part of a hemogram because it allows the quantification of different types of leukocytes and detection of morphologic abnormalities that may be indicators of pathophysiological processes. e methods of peripheral blood smear analysis used in this study mainly followed the methods of our previous study [40]. We collected blood from mice in the sham, GEG, DOX, and DOX + GEG treatment groups and conducted subsequent hematological staining with a Romanowsky stain. Blood cell morphology was evaluated in the area of the smear where the red blood cells and white blood cells were counted.

Cardiac Immunohistochemistry.
e mice in the sham, GEG, DOX, and DOX + GEG treatment groups were anesthetized and then cardiac-perfused with PBS containing 4% formaldehyde (EM grade glutaraldehyde solution, Sigma-Aldrich). We removed cardiac tissue from the mice and fixed it in 4% formaldehyde. Cardiac specimens were then embedded in paraffin and cut into 5 μm-thick tissue sections. en, tissue sections were mounted on slides for histological and immunohistochemical (IHC) analysis. e methods of cardiac immunohistochemistry used in this study mainly followed the methods of our previous study [21].

Tibia Micro-CT Measurement.
We assessed the bone density of the tibia in all mice through micro-CT measurement, which was performed by the Taiwan Mouse Clinic. A Skyscan 1076 micro-CT device (Skyscan, Aartselaar, Belgium) was used to obtain the micro-CT images of the tibias of the mice in the sham, GEG, DOX, and DOX + GEG treatment groups.
e scanning parameters were set as follows: 49 kV, 200 mA, 500 ms, and a voxel resolution of 18.27 mm. e micro-CT images were imported into CTAn software (Skyscan), and the bone volume of the tibias was calculated using ImageJ (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis.
e methods of statistical analysis used in this study mainly followed the methods of our previous study [39]. We expressed data as the mean-± standard error of the mean (SEM). Differences among the sham, GEG, DOX, and DOX + GEG treatment groups were evaluated using two-way analysis of variance (ANOVA). e Student-Newman-Keuls multiple comparison post hoc test was performed if a significant F value was obtained. Significance was defined as p < 0.05.

DPPH Free-Radical Scavenging Activity of GEG.
e free-radical scavenging activity of GEG extract at various concentrations was measured, and the results are depicted in Figure 2(a). Significant DPPH radical scavenging activity was evident at GEG concentrations of 5-20 mg/mL. Moreover, treatment with 5-20 mg/mL GEG extract yielded superior antioxidant activity (55.2%-72.7%) compared with treatment with 1 mg/mL GEG extract (10.1%). e Evidence-Based Complementary and Alternative Medicine quantified DPPH free-radical scavenging activity was similar for 10 and 20 mg/mL GEG extract; however, significant freeradical scavenging activity was observed at 20 mg/mL GEG extract treatment (p < 0.01). e HIG-82 cell viability at concentrations of 5-50 mg/mL of GEG extract without DOX treatment differed significantly from that of the control (0 mg/mL of GEG extract without DOX treatment). Moreover, treatment with 1-50 mg/mL GEG extract without DOX treatment yielded higher cell viability (115%-131%) compared with treatment with 0 mg/mL GEG extract without DOX (100%). Furthermore, our data revealed that the cell viability of HIG-82 cells was significantly reduced to approximately 56% after DOX treatment with 0 mg/mL GEG extract (p < 0.01), whereas the cell viability of DOX-treated HIG-82 cells was significantly increased to 61%-99% at GEG concentrations of 1-50 mg/ mL. e cell viability was significantly different (p < 0.01) at concentrations of 5-50 mg/mL GEG extract with DOX treatment than at concentrations of 0 and 1 mg/of GEG extract with DOX treatment.

Effect of GEG Treatment on Weight Loss in DOX-Treated
Mice.
e quantified body weights of the mice in the sham, GEG, DOX, and DOX + GEG treatment groups are presented in Figure 3(a). No significant difference in body weight was observed between mice treated with sham and

Effect of GEG Treatment on the Exercise Capacity of DOX-Treated
Mice. e quantified exhaustive swimming times of the mice in the sham, GEG, DOX, and DOX + GEG treatment groups are shown in Figure 3(b). Similar to the changes in body weight, no significant difference in exhaustive swimming time was observed between mice treated with sham and those treated with GEG (sham, 307 s vs. GEG, 320 s, p > 0.05). e exhaustive swimming time was significantly reduced in DOXtreated mice compared with that in sham-treated mice (DOX, 160 s vs. sham, 307 s, p < 0.01). However, the exhaustive swimming time was significantly increased in DOX-treated mice receiving oral GEG treatment compared with DOXtreated mice that were not treated with GEG (DOX + GEG, 292 s vs. DOX, 160 s, p < 0.01).

Effect of GEG Treatment on Subcutaneous Microcirculation in DOX-Treated
Mice. Dorsal images of subcutaneous microcirculation and quantified subcutaneous blood flow among the mice in the sham, GEG, DOX, and DOX + GEG treatment groups are shown in Figure 4(a). e subcutaneous microcirculation of mice treated with GEG was obviously enhanced but was obviously lower following DOX treatment.

Effect of GEG Treatment on Red Blood Cell Count in DOX-Treated Mice.
Peripheral blood smear analysis is routinely performed in our laboratory to evaluate myelosuppression related to dysfunction in blood cell production. We evaluated the peripheral blood smear of mice treated with sham, GEG, DOX, and DOX + GEG, and the results are presented in Figure 4(b). We observed that the number of red blood cells was obviously decreased but that the number of white blood cells was obviously increased in mice following DOX treatment. Quantification of the number of red blood cells revealed no significant difference between the mice treated with sham and those treated with GEG (sham, 9.4 × 10 6 /mm 3 vs. GEG 9.6 × 10 6 /mm 3 , p > 0.05); by contrast, the red blood cell count was significantly reduced in DOX-treated mice (sham, 9.4 × 10 6 /mm 3 vs. DOX, 4.3 × 10 6 /mm 3 , p < 0.01). Two weeks after oral GEG treatment, the number of red blood cells was significantly increased in DOX-treated mice (DOX, 4.3 × 10 6 /mm 3 vs. sham, 9.1 × 10 6 /mm 3 , p < 0.01).

Effect of GEG Treatment on Cardiac Oxidative Stress and
Inflammation in DOX-Treated Mice. We evaluated cardiac oxidative stress by using IHC staining for SOD2 in the cardiac tissues of mice treated with sham, GEG, DOX, and DOX + GEG, and the results are presented in Figure 5. SOD2 plays a major role in defense against free radicals. We observed that cardiac SOD2 expression was obviously enhanced in mice treated with GEG but was obviously reduced in mice following DOX treatment. Compared with DOXtreated mice that were not treated with GEG, those that were treated with GEG exhibited noticeably increased cardiac SOD2 expression levels. Irrespective of the presence or absence of DOX treatment, GEG can obviously relieve cardiac oxidative stress in mice. Furthermore, we evaluated inflammation through IHC staining of TNF-α in the cardiac tissue of mice treated with sham, GEG, DOX, and DOX + GEG, and the results are presented in Figure 6. TNFα is a strong proinflammatory cytokine that plays an important role in the immune system during inflammation. We observed that the cardiac TNF-α expression levels of mice treated with GEG did not exhibit obvious changes; however, the expression levels were obviously enhanced in mice following DOX treatment. Compared with DOX-treated mice that were not treated with GEG, those that were treated with GEG exhibited noticeably reduced cardiac TNF-α expression levels.

Effect of GEG Treatment on Tibia Bone Density in DOX-Treated Mice.
High-resolution microcomputed tomography was employed for cross-sectional imaging of the tibias and trabecular bones of the mice treated with sham, GEG, DOX, and DOX + GEG (Figure 7(a)). We observed that the tibia and trabecular bone density in the mice treated with sham and GEG was considerably high but obviously low in the DOX-treated mice. e density of the tibia and trabecular bone in DOX-treated mice after oral GEG treatment was obviously increased compared with that of the DOX-treated mice that were not treated with GEG. We quantified the bone density of the mice treated with sham, GEG, DOX, and DOX + GEG, and the results are presented in Figure 7(b). No significant difference in bone density was observed between the mice treated with sham and those treated with GEG (sham, 9.9% vs. GEG, 9.7%, p > 0.05). e bone density of the mice treated with DOX was significantly reduced compared with that of the mice treated with sham (DOX, 4.2% vs. sham, 9.9%, p < 0.01). However, bone loss was significantly mitigated in DOX-treated mice after GEG treatment (DOX, 4.2% vs. DOX + GEG, 7.1%, p < 0.01). Although the DOX-treated mice receiving GEG treatment could not return to their normal bone density levels (sham, 9.9% vs. GEG + DOX, 7.1%, p < 0.01), the results indicate that GEG treatment significantly increased the bone density in mice after DOX treatment.

Discussion
In the present study, we used DOX, a chemotherapeutic drug, to treat the synoviocyte cell line HIG-82 and mice to evaluate whether GEG can be used in the prevention and treatment of chemotherapy-induced side effects such as weight loss, motor disability, myocardial injury, joint 6 Evidence-Based Complementary and Alternative Medicine degeneration, and bone loss. GEG is a multicomponent formula that has been widely used in the treatment and prevention of osteoporosis for hundreds of years. GEG contains Cornu Cervi, Testudinis Plastrum, Ginseng Radix, and Lycii Fructus in specific ratios. Cornu Cervi and Testudinis Plastrum can alleviate fatigue and increase red blood cell counts and hemoglobin levels. Ginseng Radix can strengthen immunity and hematopoietic function, alleviate fatigue, and strengthen the heart. Lycii Fructus has nourishing properties and can improve eyesight, promote liver cell regeneration, increase hematopoietic function, and strengthen immunity [32]. e ancient Chinese herbal medicine book "Compendium of Materia Medica" reported that Cornu Cervi can promote bone growth and prevent osteoporosis, thereby nourishing the yang. Testudinis Plastrum can strengthen the heart, kidney, and blood, thereby nourishing the yin. If Cornu Cervi, Testudinis Plastrum, Ginseng Radix, and Lycii Fructus function as expected, people can achieve strength and vitality by consuming the four ingredients in one formula [31]. e present study compared the effects of GEG treatment on DPPH freeradical scavenging; cytotoxicity in DOX-treated synoviocytes; and weight loss, exhaustive exercise capacity, myocardial oxidative stress and inflammation, and tibia bone density in DOX-treated mice.
e major findings of this study reveal that GEG can significantly enhance DPPH freeradical scavenging (Figure 2(a)) and reduce DOX-induced cytotoxicity in synoviocytes (Figure 2(b)). In addition, the results indicate that GEG treatment can significantly mitigate weight loss (Figure 3(a)), enhance exhaustive exercise capacity (Figure 3(b)), improve blood circulation (Figure 4(a)), alleviate myocardial oxidative stress ( Figure 5) and inflammation (Figure 6), and increase tibia bone density (Figure 7) in DOX-treated mice. To the best of our knowledge, the present study is the first to conduct an evidence-based investigation of the effectiveness of  Evidence-Based Complementary and Alternative Medicine alternative therapy with the traditional Chinese medicine GEG in reversing DOX-induced weight loss, motor disability, blood circulation defects, myocardial injury, joint degeneration, and bone loss. DOX is an effective anticancer agent that inhibits cell proliferation but induces oxidative stress, ultimately leading to cell death, mainly through apoptosis [41]. Free radicals are mainly produced in the mitochondria, and they increase oxidative stress. Reactive oxygen species (ROS) comprise free radicals that can be permanently removed by a complex antioxidant system. Imbalance in the antioxidant system may cause oxidative stress and subsequent ROS overproduction. e present study demonstrated that GEG treatment significantly enhanced DPPH free-radical scavenging activity (Figure 2(a)) and alleviated myocardial oxidative stress by enhancing the cardiac expression of SOD2 ( Figure 5). Consistent with our previous report [21], we found that DOXtreated mice exhibited significantly reduced mortality rate, body weight, and cardiac function. In addition, the cardiac expression of endothelial nitric oxide synthase, SOD2, and B-cell lymphoma 2 (Bcl-2) was significantly suppressed, but the expression of TNF-α, Bcl-2-associated X protein, calpain, caspase 12, caspase 9, and caspase 3 was significantly enhanced in DOX-treated mice. ese results indicate that DOX-treated mice exhibited myocardial oxidative stress, inflammation, and apoptosis. e present study further verified that GEG treatment can mitigate weight loss (Figure 3(a)) and enhance subcutaneous microcirculation (Figure 4(a)). GEG treatment can also alleviate myocardial inflammation by reducing the cardiac expression of TNF-α ( Figure 6).
In patients with cancer undergoing chemotherapy, ameliorating chemotherapy-induced side effects such as fatigue and muscle weakness is crucial for improving patients' quality of life [42,43]. Chemotherapy-induced fatigue and muscle weakness seem to be associated with higher cancer mortality [44][45][46]. For patients undergoing chemotherapy, exercise is considered an effective treatment strategy because it can enhance muscle strength, improve quality of life, and reduce fatigue levels [47]. Additionally, the present study showed that DOX impaired the exhaustive exercise capacity of mice but that GEG treatment significantly improved the muscle strength in DOX-treated mice (Figure 3(b)). GEG has been reported to prevent and treat myelosuppression following cancer chemotherapy [35]. Blood cell deficiency caused by bone marrow suppression is one of the most serious side effects of chemotherapy. Severe bone marrow suppression and hematological toxicity after chemotherapy are major contributors to high mortality and morbidity in patients with cancer. We observed that although the number of red blood cells was obviously reduced, after 2 weeks of oral GEG treatment, the number significantly increased in DOX-treated mice (Figure 4(b)). Our results suggest that adequate exercise plus GEG treatment might be an effective therapy for alleviating chemotherapyinduced adverse side effects such as motor disability and myelosuppression.
Osteoarthritis is the most prevalent form of joint disease and often causes falls, disabilities, and dependency in older people [28,29]. In vitro experiments with synoviocyte HIG-82 cells isolated from soft tissue lining the knee joints of rabbits are recommended to evaluate the pathophysiology of various arthritides [48]. Our data showed that the viability of HIG-82 cells was significantly reduced to approximately 56% after DOX treatment, whereas the viability of DOX-treated HIG-82 cells was significantly increased to 61%-99% after GEG treatment (Figure 2(b)). e results indicate that DOX treatment may cause synoviocyte damage and even lead to arthritides, whereas GEG treatment may reduce DOX-induced cytotoxicity and facilitate synoviocyte proliferation. In addition, osteoporosis has become a public health concern as a result of the high risk of fractures and subsequent complications that it incurs [30]. Children receiving DOX chemotherapy may experience bone damage in the form of reduced adult height and increased fracture risk [22]. Patients with cancer receiving DOX chemotherapy may exhibit low bone mineral density and significant bone loss [23]. DOX chemotherapy may cause a 60% reduction in bone formation in normal rats [24,25] and reduce trabecular bone volume and cortical bone thickness in rabbits [26]. Our results reveal that the density of the tibia and trabecular bone was obviously decreased in mice treated with DOX, whereas it was obviously increased in DOX-treated mice treated with oral GEG (Figure 7). GEG primarily originates from processed tortoise shells and antlers and has been widely used in China for the treatment and prevention of osteoporosis for hundreds of years with minimal side effects [31]. Our results suggest that DOX treatment may cause osteoporosis in mice but that GEG treatment can relieve this osteoporosis. Although bone density of GEG group mice seems to be lower than that of the sham group, there is no significant difference between sham and GEG group mice (Figure 7(b)). We were worried that longterm oral administration of GEG may reduce bone density. Until now, there is no evidence that long-term oral administration of GEG may cause a crisis of osteoporosis. e mechanisms by which GEG treatment relieves osteoporosis and osteoarthritis remain unclear. erefore, further clinical trials are warranted to verify the benefits of GEG treatment in chemotherapy-treated patients.

Conclusions
e study findings reveal that DOX chemotherapy can induce cytotoxicity in synoviocyte HIG-82 cells and cause motor disability, myocardial oxidative stress and inflammation, and bone loss in mice. GEG treatment can significantly enhance DPPH free-radical scavenging activity and alleviate DOX-induced cytotoxicity in synoviocyte HIG-82 cells. Moreover, oral GEG treatment for 2 weeks can significantly mitigate weight loss, enhance exhaustive exercise capacity, alleviate myocardial oxidative stress and inflammation, and strengthen the tibia in DOX-treated mice. us, GEG treatment is suggested to be an alternative therapy for alleviating chemotherapy-induced adverse side effects.

Data Availability
Blood biochemical analysis, DPPH assay, MTT assay, immunohistochemistry, and micro-CT measurement data used to support the findings of this study are included within the article and available from the corresponding author upon request.

Conflicts of Interest
ere are no conflicts of interest in this study.