Effect of salmon calcitonin combined with calcium antagonist on blood calcium and phosphorus ion concentration in osteoporosis rats

Abstract

Background

Our research aims to explore the effect of salmon calcitonin (sCT) combined with three calcium antagonists (CCA), verapamil, nifedipine and diltiazem, on calcium and phosphorus ion metabolism in the blood of osteoporotic female rats following ovarian removal. To explore a method of combination medication suitable for postmenopausal women with osteoporosis.

Methods

We first carried out the combined drug experiment on rats, and then carried out the combined drug experiment on osteoporosis rats. Blood samples were collected at 1, 2, 4, 8 and 12 h post-administration, and then we measured the concentration of calcium and phosphorus ions in serum.

Results

Osteoporotic rats were divided into eight groups, with 6 rats in each group, with an average weight of 350 g. By studying changes in the concentration of calcium and phosphorus ions in the blood over a period of time, we found that the effect of sCT combined with nifedipine and verapamil in reducing serum calcium and phosphorus was better than that of single drug; the effect of sCT combined with diltiazem in reducing serum calcium was better than that of single drug, and the effect of reducing blood phosphorus was not as good as that of single drug.

Conclusions

We have studied and found that the group of sCT combined with nifedipine group has the most significant effect on reducing calcium and phosphorus in the blood of osteoporotic rats. The effect was significantly better than salmon calcitonin alone. That provided a new medication method for postmenopausal women with osteoporosis.

Clinical trial number.

Not applicable.

Background

Osteoporosis, a genetic ailment, exhibits a profound link between bone mass and genetic factors. The susceptibility to osteoporosis is notably heightened, especially among daughters, if their mothers have experienced severe osteoporosis. Multiple factors, such as ethnicity, exercise routines, dietary habits, and daily lifestyles, further contribute to the complex web of osteoporosis causation. This study centers on postmenopausal women afflicted with both osteoporosis and hypertension—a study domain positioned within the broader scope of treating hereditary conditions. The prevalence of osteoporosis has surged in recent times, propelled not only by advancing age but also by persistent influences from the environment and diet. Notably, osteoporosis has surged among individuals over 60, predominantly women, amplifying the rates of disability and mortality [1]. The specter of fractures looms prominently due to severe osteoporosis. One pivotal cause of postmenopausal osteoporosis in women is the occurrence of minor traumatic fractures. During bone metabolism, osteoclasts (OCs) adhere to trabecular bone surfaces, resorbing bone matrix and releasing calcium into the surrounding milieu. Simultaneously, osteoblasts (OBs) secrete bone matrix and osteocalcin, fostering new bone formation. During growth stages, both actions intensify, but osteoporosis disrupts this balance, reducing osteoblast activity and increasing osteoclast activity [2]. Remedies for osteoporosis must not solely curb bone resorption but also promote bone formation [3].

Salmon calcitonin (sCT), a 32-amino-acid polypeptide hormone, intricately manages calcium and phosphorus metabolism. It dually restrains osteoclast activity and kindles osteoblast formation, thwarting bone decalcification and averting calcium depletion. It addresses hypercalcemia, osteoporosis, diabetes, gastric ulcer, acute pancreatitis, deformable bone disease (Paget’s disease), painful neurodystrophy (Sudeck’s disease) and malignant osteolysis. Available in nasal spray, suppository, and injection forms, sCT is often paired with conventional drugs in clinical practice, yet its superiority over monotherapy remains unverified [1].

Debuting in 1975, nifedipine emerges as a blood pressure regulator that augments endothelial functionality [4]. It curbs Ca²⁺ influx, relaxing vascular smooth muscles, expanding coronary arteries, bolstering coronary blood flow, and enhancing myocardial tolerance to ischemia [5]. In the same vein, verapamil, a pioneer antiarrhythmic, anti-angina, and antihypertensive phenylalkylamine [6], acts as an L-type calcium channel antagonist, diminishing intracellular calcium overload [7], regulate calcium influx in the membrane of myocardial conduction cells, myocardial contractile cells, and arterial vascular smooth muscle cells. Diltiazem can bind to slow calcium channels in phase 2 of the action potential, prevent calcium ions from flowing into cells, dilate coronary arteries and peripheral blood vessels, block potassium ion outflow and sodium ion influx, prolong the effective refractory period of the atrioventricular node, dilate arterial vessels, reduce peripheral vascular resistance, and eliminate coronary vasospasm. This can increase blood supply to the coronary artery and improve blood supply to the myocardium.

Osteoporosis will lead to an increase in the concentration of calcium and phosphorus ions in the blood. When the concentration of these ions reaches an abnormal value, it will have a significantly effect on blood pressure. For example, when the calcium content in the blood exceeds the standard, it will increase the brittleness of arteries and lead to vascular hardening. In the long run, due to the increase of blood pressure on the arterial wall, it will cause blood pressure to rise. Even if there is an underlying disease of hypertension, it can increase the risk of high blood pressure. Therefore, we speculated that antihypertensive drugs may improve the effectiveness of treating osteoporosis, and the study object chosen was calcium antagonists. In this study, the possible mechanism is that calcium antagonists inhibit the flow of calcium ions into cells, this keeps calcium ions in the blood and makes salmon calcitonin more effective in depositing calcium ions on bones, which act synergistically. The purpose of the study is to explore a more effective combination medication method for the treatment of osteoporosis.

Methods

Materials

Laboratory animal

A total of 124 female SD rats (SPF grade) were purchased from Jinan Pengyue Experimental Animal breeding Co., Ltd. Out of these, 66 were used in the drug experiment of normal rats, 53 were used to establish the osteoporosis model, and 5 were used to verify the success of the osteoporosis model as a sham operation group. The calcium content of the feed used is 1.0–1.8%, and the total phosphorus content is 0.6–1.2%. This study has passed the review of the Experimental Animal Ethics Committee of Shandong Second Medical University, acceptance number (2021 SDL231).

Main reagents and drugs

Salmon calcitonin injection (Miacalcic), Nifedipine Sustained release Tablets (II) (Dezhou Bocheng Pharmaceutical Co., Ltd.), Verapamil hydrochloride tablets (Tianjin City Central Pharmaceutical Co., Ltd.), Diltiazem hydrochloride Sustained Release Capsules (II) (Tianjin Biantian Pharmaceutical Co., Ltd.), 0.9% physiological saline.

Methods

The method of combined use of drugs in normal rats

The average age of normal rats is 5–6 months, and were divided into 11 groups with 6 rats in each group. The experimental rats were adaptively fed for a week, took water and diet freely, were weighed before the experiment, and the average weight was about 360 g. They were fasted for 12 h before the experiment, with no control over water intake. The SCT injection (1 ml: 50 IU) was diluted tenfold with 0.9% normal saline and was now being used. According to the conversion coefficient of rat weight, the injection volume is about 0.5 ml and the volume of intragastric administration is 2 ml. The concentration of low-dose group and high-dose group was half and double that of middle-dose group, respectively. Group 1 was the blank control group, group 2 was the sCT group, group 3 was the sCT plus nifedipine low-dose group, group 4 was the sCT combined with nifedipine medium-dose group, group 5 was the sCT plus nifedipine high-dose group, group 6 was the sCT plus verapamil low-dose group, group 7 was the sCT plus verapamil medium-dose group, group 8 was the sCT plus verapamil high-dose group, group 9 was the sCT combined with diltiazem low-dose group. There were 10 groups of sCT combined with diltiazem at medium dose, and 11 groups of sCT combined with diltiazem at high dose. Salmon calcitonin was injected intraperitoneally, and calcium antagonist was administered intraperitoneally, once at 7:30 a.m. The specific doses and modes of administration are shown in Table 1.

Table 1.

Drug administration mode and dose in different groups of rats

Group	Intragastric sample	Gavage dose	Injection sample	Injection dose
Group 1	Normal saline	2 mL	Normal saline	0.5 mL
Group 2	Normal saline	2 mL	sCT	0.5 mL
Group 3	0.36 mg/mL Nifedipine	2 mL	sCT	0.5 mL
Group 4	0.73 mg/mL Nifedipine	2 mL	sCT	0.5 mL
Group 5	1.46 mg/mL Nifedipine	2 mL	sCT	0.5 mL
Group 6	2.188 mg/mL Verapamil	2 mL	sCT	0.5 mL
Group 7	4.375 mg/mL Verapamil	2 mL	sCT	0.5 mL
Group 8	8.75 mg/mL Verapamil	2 mL	sCT	0.5 mL
Group 9	0.82 mg/mL Diltiazem	2 mL	sCT	0.5 mL
Group 10	1.64 mg/mL Diltiazem	2 mL	sCT	0.5 mL
Group 11	3.28 mg/mL Diltiazem	2 mL	sCT	0.5 mL

After the intragastric administration, blood samples were collected from tail vein at 1, 2, 4, 8 and 12 h, respectively, ensuring a blood volume of not less than 0.5 ml each time. Once the blood collection was completed, the samples were stored at 37 ℃ for 30 min, followed by cooling to room temperature. Subsequently, they were centrifuged at 1000 g for 20 min, and the supernatant was collected and stored at − 20 ℃. The contents of blood glucose, calcium and phosphorus in serum were detected by automatic biochemical analyzer.

Method of establishing female rat model of osteoporosis

Rats aged 6–8 weeks were selected, and were anesthetized by intraperitoneal injection of 2% barbital sodium solution, with an injection volume of 0.2 ml per 100 g. After achieving anesthesia with no blink reflex, the rats were fixed in the ventral position and exposed to the operation site at the intersection of the axillary midline and 1–2 cm lateral from the spine on both sides of the rats. After disinfecting the towels with 75% alcohol, the skin was cut, back muscles and sarcolemma in turn. The white adipose tissue was pull out of the incision gently, the adipose tissue was separated, and the pink ovaries can be seen. The fallopian tube at the lower end of the ovary was ligated, then the ovary was removed cleanly. Continue to remove the ovary on the other side, the inner muscle and outer skin were sutured in turn, and disinfect it with iodine tincture. The sham operation group only exposed the ovary but did not undergo resection, and the rest of the operation was the same as that of the model group. After the operation, the animals were fed in cages and received normal feeding. Penicillin was injected intramuscularly 3 days after modeling, with a dose of 800,000 units per day. The animals resumed feeding for 12 weeks after the operation. Rats were kept in a ventilated and dry environment at room temperature. Five rats in the sham operation group and five rats in the model group were examined by Micro CT was carried out to verify whether the model was successfully established. The data of the sham operation group were averaged, and the model group selected the data of the two most typical rats for display.

Micro-CT method

Sample processing

1. Place the prepared tissue specimens in 4% polyformaldehyde solution and fix them for 24–48 h.

2. The removed specimens were washed three times with PBS buffer.

3. Place the specimen in 75% alcohol solution and store it at 4 ° C for later use.

Scanning analysis

Fixed and preserved tissue samples were scanned using the SkyScan1276 Small Animal Micro-CT Scanning Image System of Bruker Company in Germany, with a resolution of 18 um. Systematic analysis software was used to analyze the microstructure parameters of bone tissue in the target area, and the analysis range of all specimens remained consistent.

Experimental method of combined administration of drugs in osteoporotic model rats

Intervention began 15 weeks after ovariectomy. The osteoporosis rat’s average age is 5–6 months, and were weighed before the experiment, the average weight was 350 g. The osteoporosis rats were divided into 8 groups with 6 rats in each group. The determination of the sample size is to increase the sample size as much as possible to make the results more accurate if funding permits, and the number of each group was consistent with the previous normal rat experiment. Before the experiment, the model rats fasted for 12 h, had unrestricted access to drinking water, and their average body weight was calculated to be about 350 g. According to the drug instructions and the conversion coefficient of rat body weight, the optimal doses were determined as follows: group 1 served as the blank control group, group 2 received sCT, group 3 received nifedipine, group 4 received verapamil, group 5 received diltiazem, group 6 received sCT combined with nifedipine, group 7 received sCT combined with verapamil, and group 8 received sCT combined with diltiazem. Salmon calcitonin was injected intraperitoneally, and calcium antagonist was administered intraperitoneally, once at 7:30 a.m. The specific doses and administration methods are shown in Table 2.

Table 2.

Drug administration mode and dose in different groups of osteoporotic model rats

Group	Intragastric sample	Gavage dose	Injection sample	Injection dose
Group 1	Normal saline	2 mL	Normal saline	1 mL
Group 2	Normal saline	2 mL	sCT	1 mL
Group 3	0.73 mg/mL Nifedipine	2 mL	Normal saline	1 mL
Group 4	4.375 mg/mL Verapamil	2 mL	Normal saline	1 mL
Group 5	1.64 mg/mL Diltiazem	2 mL	Normal saline	1 mL
Group 6	0.73 mg/mL Nifedipine	2 mL	sCT	1 mL
Group 7	4.375 mg/mL Verapamil	2 mL	sCT	1 mL
Group 8	1.64 mg/mL Diltiazem	2 mL	sCT	1 mL

Blood samples were collected from the tail vein at 1, 2, 4, 8 and 12 h after intragastric administration. The blood samples were collected in not less than 0.5 ml each time. After the whole blood was placed at room temperature for 30 min, the serum was centrifuged at 4 ℃ and 3000 rpm for 10 min. The serum was removed into a new centrifuge tube and placed at 4 ℃ overnight. The concentration of calcium and phosphorus in blood was detected by an automatic biochemical analyzer the next day.

Conversion of drug dosage in rats

The average weight of the rat was 0.35 kg. According to the dose table of human and animal body surface area ratio in “Pharmacological Experimental Methodology”, the equivalent dose ratio between rats and humans is calculated to be about 6.25. It can be concluded:

Diltiazem hydrochloride: adults take 90 mg orally daily, body weight 60 kg, dose 1.5 mg/kg; rat dose 1.5*6.25 = 9.375 mg/kg; gavage dose 3.28 mg, volume 2 mL, configuration concentration is 1.64 mg/mL.

Nifedipine: adults take 90 mg orally daily, body weight 60 kg, dose 0.67 mg/kg; rat dose 0.67*6.25 = 4.167 mg/kg, gavage dose 1.46 mg, volume 2 mL, configuration concentration is 0.73 mg/mL.

Verapamil hydrochloride: adults orally 240 mg/day, body weight 60 kg, dose 4.0 mg/kg; rat dose 4.0*6.25 = 25.00 mg/kg, gavage dose 8.75 mg, volume 2 mL, configuration concentration is 4.375 mg/mL.

sCT injection: adults injection 100 IU/day, average body weight 60 kg, dose 1.67 IU/kg; rat dose 1.67*6.25 = 10.44 IU/kg, injection dose 3.654 IU, volume 1 mL, configuration concentration is 3.654 IU/mL. Dilute 50 IU/mL of the drug to 3.654 IU/mL.

Data analysis and statistics

In this study, the data of blood glucose, blood phosphorus and blood calcium concentration were analyzed by GraphPrism9.0.0 software for one-way analysis of variance (ANOVA). Independent sample t tests were used to analyze the data for each individual group. The statistical results indicated a significant difference based on a bilateral test (P < 0.05).

Results

Experimental results of normal rats

Effects of sCT combined with nifedipine, verapamil and diltiazem on blood glucose in normal rats

As can be seen from Fig. 1A, the blood glucose concentration of sCT combined with nifedipine in low and middle dose groups decreased first and then increased, while that in the high dose group showed an overall downward trend. The blood glucose concentration in the low, middle and high dose groups was the highest at 4 h (9.31 mmol/L in the middle and low dose groups, 8.95 mmol/L in the high dose group), and the lowest in the low and middle dose groups at 8 h (low dose group: 5.01 mmol/L).(middle dose group: 3.69 mmol/L). The blood glucose concentration of the high dose group reached the lowest value at 12 h, the blood glucose concentration of the three low, middle and high nifedipine groups was lower than that of the blank control group and sCT group at 1 h to 12 h after treatment, and the hyperglycemic effect of the middle dose group was the weakest. As can be seen from Fig. 1B, the blood glucose concentration of the low, middle and high dose groups of sCT combined with verapamil initially decreased and then increased. The blood glucose concentration of the high and low dose groups was the highest at 2 h (high dose group: 8.07 mmol/L, low dose group: 6.92 mmol/L), while the middle dose group reached the highest value of 7.62 mmol/L at 1 h. The blood glucose concentration of all three groups was the lowest at 8 h (low dose group: 2.99 mmol/L, middle dose group: 2.99 mmol/L). The high dose group: 4.61 mmol/L), the blood glucose concentration of the middle and low dose groups was close to that of the blank control group, but lower than that of the sCT group. The blood glucose concentration of the high dose group was close to that of the sCT group and higher than that of the blank control group from 1 to 4 h, and higher than that of the blank control group and sCT group from 4 to 12 h after treatment. The hyperglycemic effect of the middle dose group was the weakest. As can be seen from Fig. 1C, the overall trend of blood glucose concentration in the sCT combined with diltiazem low-dose group, middle-dose group and high-dose group was first decreased and then increased. The blood glucose concentration in the low-dose group reached the highest 8.24 mmol/L at 1 h, while that in middle-dose group and high-dose group reached the highest at 4 h (middle-dose group: 7.30 mmol/L, high-dose group: 7.54 mmol/L). The blood glucose concentration in all three groups was the lowest at 8 h (low-dose group: 2.79 mmol/L, middle-dose group: 4.90 mmol/L, high dose group: 4.48 mmol/L). The overall blood glucose concentration of the low dose group is lower than that of blank control group, while the overall blood glucose concentration of the middle and high dose group is higher than that of the blank control group. Therefore, the hypoglycemic effect of the low dose group is the weakest.

Fig. 1.

Effects of sCT combined with nifedipine (A), verapamil (B) and diltiazem (C) on blood glucose in normal rats

One-way ANOVA showed that there was a significant difference between the high-dose sCT combined with nifedipine group and the blank control group, with statistical significance (P < 0.05). There were no significant differences in the other groups. The results showed that when the dose of nifedipine exceeded 2.92 mg, the blood glucose level of sCT combined with nifedipine increased significantly, and the blood glucose concentration reached the highest level at 4 h after treatment, reaching 9.35 mmol/L. The hyperglycemic effect of the other groups was not significant.

Effects of sCT combined with nifedipine, verapamil and diltiazem on serum calcium in normal rats

As can be seen from Fig. 2A, the serum calcium concentration in the middle and high dose groups of sCT combined with nifedipine continuously decreased and reached the lowest at 12 h (middle dose group 12 h: 1.98 mmol/L, high dose group 12 h: 1.91 mmol/L). There was no significant difference between the two groups. The low dose group showed a downward trend at 0 ~ 8 h, and increased slowly at 12 h. The concentration of serum calcium in the low dose group was lower than that in the blank control group, sCT group, middle and high dose groups, and the ability to reduce serum calcium was stronger. As can be seen from Fig. 2B, the serum calcium concentration in the middle, low and high dose groups of sCT combined with verapamil initially decreased and then increased, and all reached their lowest level at approximately 4 h after administration (low dose group: 1.89 mmol/L, middle dose group: 2.02 mmol/L, high dose group: 1.95 mmol/L). The serum calcium concentration in the high dose group began to rise after 4 h, and continued to decrease in the middle and low dose groups at 4 h and 8 h after treatment. The total serum calcium concentration in the low dose group was lower than that in the blank control group, sCT group, middle and high dose groups, and the low dose group had a stronger ability to reduce blood calcium. As can be seen from Fig. 2C, there was little difference in the change curve among the middle, low and high dose groups of sCT combined with diltiazem. The serum calcium concentration of the three groups was lower than that of the blank control group and sCT group from 1 to 4 h, and lower than that of the blank control group and higher than that of the sCT group at 4 h after treatment. The serum calcium concentration of the three groups reached its lowest point at approximately 4 h after treatment, and the value was 2.01 mmol/L.

Fig. 2.

Effects of sCT combined with nifedipine (A), verapamil (B) and diltiazem (C) on serum calcium in normal rats

There were significant differences between the middle, high and low-dose groups of sCT combined with nifedipine, the blank control group and the sCT group. There were significant differences between the middle, high and low dose groups of sCT combined with verapamil, the blank control group and the sCT group. SCT combined with diltiazem low, middle and high dose group, the blank control group and the sCT group only showed a significant difference within 4 h. For normal rats, the low-dose group of sCT combined with nifedipine and verapamil showed a significant calcium-lowering ability, while sCT combined with diltiazem only had a significant calcium-lowering ability at 0 ~ 4 h.

Effects of sCT combined with nifedipine, verapamil and diltiazem on serum phosphorus in normal rats

It can be seen from Fig. 3A that the blood phosphorus concentration of the low-dose group, middle-dose group and high-dose group of sCT combined with nifedipine initially increased and then decreased from 1 to 12 h after administration. The blood phosphorus concentration reached its lowest at 1 h (low-dose group: 2.12 mmol/L, middle-dose group: 2.12 mmol/L, and high-dose group: 2.40 mmol/L), and the highest at 8 h (low-dose group: 3.82 mmol/L, middle-dose group: 4.12 mmol/L, and high-dose group: 4.12 mmol/L). The overall serum phosphorus concentration of sCT combined with nifedipine group was higher than that of sCT group, and the ability to reduce blood phosphorus was not as effective as that of sCT group. As can be seen from Fig. 3B, the blood phosphorus concentration of the three dose groups receiving sCT combined with verapamil initially increased and then decreased. The blood phosphorus concentration of the middle and low dose groups reached the lowest value 2.05 mmol/L after 2 h, and the blood phosphorus concentration of the high dose group reached its lowest value 2.00 mmol/L at 4 h. The whole blood phosphorus concentration of the three groups was lower than that of the sCT group. Figure 3C shows that the blood phosphorus concentration of the three dose groups receiving sCT combined with diltiazem initially increased and then decreased from 1 to 12 h. The blood phosphorus concentration was the lowest in middle dose group and high dose group at 1 h (middle-dose group: 1.76 mmol/L, high-dose group: 1.67 mmol/L). Among the three dose groups, only the whole blood phosphorus concentration of high dose group was lower than that of sCT group.

Fig. 3.

Effects of sCT combined with nifedipine (A), verapamil (B) and diltiazem (C) on serum phosphorus in normal rats

Regardless of the combination was used, there were no significant difference between the three dose groups and the blank control group and the sCT group (P > 0.05). For normal rats, there was minimal distinction in the regulation of blood phosphorus concentration between the combination group and the single drug group. The effect of the high-dose group exhibited a more effective impact than that of the middle and low-dose groups, yet not as good as that of sCT alone.

Results of establishment of osteoporosis model rats

The results of evaluating the success of the osteoporosis model in rats are shown in Table 3. According to the Micro CT images of the anterior, posterior, left and right, and the section from the right side (Fig. 4) of the femur of rats, it is evident that the femur surface pores in the model rat group are significantly more abundant than those in the sham operation group. In addition, the bone cortex becomes thinner and the brightness increases. The results of bone mineral density indicated that the bone mineral density of model 1 and 2 was 0.10281 g/cm³ and 0.4317 g/cm³, respectively, which was significantly lower than that of sham operation group (0.3404 g/cm³). The bone volume percentage of model 1 and 2 was 4.99746% and 3.10596%, respectively, which was much lower than that of sham operation group (28.05446%). The open voids were 95.00254% and 96.89404%, respectively, which were higher than that of sham operation group (71.94532%), and the connectivity was 132,93, respectively. All of them were lower than those in the sham operation group (586), and the bone trabeculae decreased significantly. The results showed that the successful establishment of the rat model of osteoporosis.

Table 3.

Relevant data of osteoporosis rat model establishment

	Model-1	Model-2	Sham operation group
Bone mineral density (BMD)	0.10281	0.04317	0.34040
Tissue volume (mm3)	10.52787	9.94303	7.65143
Bone volume (mm3)	0.52613	0.30883	2.14657
Percent bone volume (%)	4.99746	3.10596	28.05446
Tissue surface (mm2)	34.42139	33.31831	27.48982
Bone surface (mm2)	20.35785	17.11475	72.91029
Intersection surface (mm2)	2.04296	1.23284	8.19927
Volume of open pore space (mm3)	10.00175	9.63420	5.50485
Open porosity(%)	95.00254	96.89404	71.94532
Connectivity (Conn)	132	93	586
Connectivity density (1/mm3)	12.53814	9.35328	76.58695
Trabecular number (1/mm)	0.52760	0.43925	2.78215

Fig. 4.

Micro-CT image comparison: Each row is the same bone, and from left to right are the front, left, back, right and section of the bone. A is sham operation group, B is model group 1, and C is model group 2

Experimental results of combined administration of drugs in osteoporotic model rats

Effects of sCT combined with nifedipine, verapamil and diltiazem on blood calcium in osteoporotic rats

As can be seen from Fig. 5A, the serum calcium concentration in nifedipine group remains relatively stable, ranging from 2.29 to 2.44 mmol/L. In sCT combined with nifedipine group, the serum calcium concentration first decreased and then increased, reaching its lowest value of 1.76 mmol/L at 8 h. The serum calcium concentration of sCT combined with nifedipine group was significantly lower than that in nifedipine group and sCT group from 1 to 12 h after treatment, and there was significant difference among sCT combined nifedipine group, blank control group, sCT group and nifedipine group (P < 0.0001). It can be seen from Fig. 5B that the serum calcium concentration in verapamil group tends to be stable within the range of 2.30–2.42 mmol/L, and the blood calcium concentration in combination group initially decreases and then increases. This effect is most pronounced at 8 h after treatment, with the lowest value recorded at 1.80 mmol/L. Notably, this value is lower than that in verapamil group (lowest at 8 h, 2.30 mmol/L) and sCT group (lowest at 8 h, 1.86 mmol/L). Significant differences are observed among the sCT combined with verapamil group, blank control group, sCT group and verapamil group (P < 0.0001). From Fig. 5C, it is evident that the overall serum calcium concentration in diltiazem group tends to be stable within the range of 2.26–2.37 mmol/L, the serum calcium concentration in the combination group decreased at first and then increased, and decreased to the lowest value 1.76 mmol/L at 8 h. The overall serum calcium concentration in the combined group was lower than that in the sCT group and diltiazem group. There were significant differences among sCT combined with diltiazem group, blank control group, sCT group and diltiazem group (P < 0.05).

Fig. 5.

Effects of sCT combined with nifedipine (A), verapamil (B) and diltiazem (C) on serum calcium in osteoporotic rats

Effects of sCT combined with nifedipine, verapamil and diltiazem on blood phosphorus ion in osteoporotic rats

As demonstrated in Fig. 6A, the blood phosphorus concentration of the sCT combined with nifedipine group decreased steadily and continuously within 12 h, and the ability to reduce blood phosphorus was the strongest between 4–12 h. The blood phosphorus concentration reached the lowest value of 1.97 mmol/L at 12 h, and the whole blood phosphorus concentration was significantly lower than that of both the sCT group and the nifedipine group. The results of the one-way ANOVA showed that there were significant differences among the sCT combined with nifedipine group, the blank control group, the sCT group and the nifedipine group (P < 0.05). As can be seen from Fig. 6B, the blood phosphorus concentration of the sCT combined with verapamil group exhibited an overall downward trend, reaching its lowest point at 12 h after treatment, measuring at 1.89 mmol/L. The whole blood phosphorus concentration was lower than that of sCT group and verapamil group. There was significant difference between sCT combined with the verapamil group, the blank control group, the sCT group and the verapamil group (P < 0.05). As can be seen from Fig. 6C, the serum phosphorus concentration of the sCT combined with diltiazem group exhibited a continuous decrease, reaching its lowest value of 2.74 mmol/L at 12 h. The blood phosphorus concentration of combination group was nearly identical to that of the diltiazem group but higher than that of the sCT group, the effect of combination is not as good as that of sCT alone. There were significant differences among the sCT combined with the diltiazem group, the blank control group, the sCT group and the diltiazem group (P < 0.05).

Fig. 6.

Effects of sCT combined with nifedipine (A), verapamil (B) and diltiazem (C) on serum phosphorus in osteoporotic rats

Discussion

Under the dual influence of environmental factors and social pressure, more and more women in China are experiencing the phenomenon of early menopause, leading to premature suffering from postmenopausal osteoporosis. At the same time, these female patients often present accompanying conditions, such as hypertension, angina pectoris, coronary heart disease, and other cardiovascular and cerebrovascular diseases, which is a very common phenomenon in today's society. Therefore, it can be seen that many patients with osteoporosis are also taking antihypertensive drugs while taking drugs to treat osteoporosis. After speculation, we believe that the combination of calcium antagonists and salmon calcitonin will improve the treatment of osteoporosis, so we conducted this study.

The method of combining drug use has become an indispensable strategy to overcome the drug resistance of antibiotics and anticancer drugs. According to the fact that the combined effect of drugs can be greater than, equal to or less than the sum of the actions of individual drugs, the combined effects of drugs are usually divided into synergistic, additive and antagonistic effects. In this study, we used the combination of drugs to treat osteoporotic rats. When comparing the combination of drugs with single administration, the effect of reducing serum calcium and phosphorus was found to be more significant. Our experiment on osteoporosis rats was divided into eight groups. The blank control group was established to compare with other groups to determine whether the concentration of serum calcium and phosphorus changed after using drugs alone or in combination. The calcium antagonist group and sCT group were set up to compare with the combination group and to assess the advantages and disadvantages of the combination group and the single group. Combination therapy is a strategy to overcome drug resistance and has made a great contribution to the prevention and treatment of diseases, such as HIV, tuberculosis and cancer. Considering that most diseases and their drug resistance are multifactorial, multiple drugs targeting multiple components are less likely to develop resistance than using single drugs targeting multiple components. For example, in patients with BRAFv600-mutated melanoma, both mitogen-activated protein kinase (MEK) and B-Raf proto-oncogene serine/threonine protein kinase (BRAF) are targeted, not just MEK or BRAF. The main focus of drug combinations is the use of discriminative models to identify synergistic or antagonistic drugs for specific diseases [8]. Regarding the combination of drugs, there have been long-standing studies that have demonstrated its advantages. Some researchers have used simvastatin/ezetimibe or placebo. The simvastatin/ezetimibe group reduced the risk of major atherosclerotic events by 17% compared to the placebo group. The addition of ezetimibe to statins can lead to a gradual decrease in LDL-C levels without increasing the dose of statins. This proves that combining statins with ezetimibe is better than using a single drug [9]. Compared with previous studies, the combination of drugs in this study showed a synergistic effect and improved the treatment effect on the disease. In some cases, the use of a single drug can limit the efficacy of the drug and the resistance of the disease to the drug. It has been proved that the combination of drugs can improve the therapeutic effect, reduce the dosage of drugs, overcome drug resistance and reduce side effects. One of the uses of drug combinations that cover a large number of people is the treatment of chronic diseases. Generally speaking, patients with chronic diseases are typically older than general population. They suffer from a variety of diseases, and require two or more doses of drugs every day. This type of combination is intended to cover all therapeutic targets to reduce missed effects. Cancer treatment is also an arduous task that needs the combination of multiple drugs for resolution. Determining an effective drug combination is essential for finding an effective treatment for drug-resistant cancers. Recently, regulatory authorities have regarded the combination of drugs as a new type of special drug to treat a variety of cancers [10].

The combination of drugs effectively relieved the symptoms of patients with osteoporosis, inhibited bone resorption and reduced the concentration of calcium and phosphorus in the blood, which was consistent with the results obtained in this experiment. Compared with the blank group, the levels of serum calcium and phosphorus in the combination group decreased significantly. Previous studies have shown that calcium (Ca), magnesium (Mg) and calcium/magnesium ratio are associated with inflammation and metabolic disorders. In addition, several studies have shown that low Ca and Mg intake is associated with an increased risk of non-alcoholic fatty liver [11]. Calcium is an important mineral necessary to support the build and maintain bones and teeth. The human body gets the calcium it needs in two ways, one is to eat foods or supplements containing calcium, and the other is to extract calcium from bones. When the concentration of calcium in the blood decreases due to insufficient food intake, calcium is released from the bone to become part of the blood; therefore, osteoporosis is observed. It is speculated that there is a certain relationship between the content of calcium in blood and osteoporosis [12]. Hypophosphatemia can lead to ATP consumption. As a result, it causes organ dysfunction, especially muscle weakness [13].

The sCT used in this experiment is a very effective drug for osteoporosis. It can inhibit the activity of osteoclasts in patients with osteoporosis, reduce bone resorption, increase the activity of osteoblasts, increase bone formation, effectively reduce the concentration of calcium in patients' blood, and mainly participate in the plasma metabolism of calcium and phosphorus. Moreover, sCT can be used to treat hypercalcemia, Paget's disease, osteoporosis, diabetes, gastric ulcer, acute pancreatitis and other diseases [14, 15]. Simultaneously, sCT can relieve postoperative pain and promote fracture healing. It has been widely used by orthopedic surgeons as an auxiliary method for fracture treatment and as a therapeutic agent for patients with bone mineralization/demineralization disorders [16]. Because many osteoporosis drugs are not effective, researchers have found that sCT can be used to treat osteoporosis, which can not only reduce the pain of osteoporosis patients, but also effectively reduce the occurrence of adverse reactions. Currently, the main dosage forms of sCT are injections, nasal sprays and suppositories. In addition, oral preparations are still under development. Furthermore, some scholars speculate that oral preparations may be more effective than other dosage forms in some aspects [1, 17]. Because calcitonin comes from salmon, patients who are allergic to fish may be prone to hypersensitivity [18]. The activity of sCT is not sustained long enough after stopping medication, and it may be lost quickly [19]. At present, there is no way to improve it, so patients with osteoporosis need to take drugs for life.

The experimental results were consistent with expectations. The combination of drugs adjusted both blood calcium and blood phosphorus concentrations to a certain extent. The use of salmon calcitonin in combination with calcium antagonists has a better therapeutic effect on osteoporosis than salmon calcitonin alone, which greatly increases the possibility of the previously speculated mechanism. Calcium antagonists prevent calcium ions from entering cells, and patients with osteoporosis have a higher blood calcium concentration. This causes calcium ions to be retained in the blood. The two drugs act synergistically to deposit more calcium ions on the bones. This provides a more effective clinical medication reference for patients with osteoporosis. The limitation of this study is that no blood pressure monitoring was carried out, and it is not known whether clinical application will affect blood pressure. Future research needs to consider multiple influencing factors. While studying the effect of combination medication on osteoporosis, also monitors the impact of this method on blood pressure.

Conclusion

Within 12 h after treatment, the results showed that the effect of salmon calcitonin combined with nifedipine in lowering blood calcium and blood phosphorus was not only better than the single drug, but also more significant than the other two combination methods. Therefore, we think that the sCT combined with nifedipine has the most significant effect on reducing calcium and phosphorus in the blood of osteoporotic rats, and was more suitable for clinical application.

Abbreviations

Ca

Calcium

SPF grade

Female SD rats

Mg

Magnesium

MEK

Mitogen-activated protein kinase

ocs

Osteoclasts

obs

Osteoblasts

sCT

Salmon calcitonin

Author contributions

C.J.: Formal analysis, software, data curation, project administration, methodology, writing—original draft. W.J.: Project administration, data curation. J.W.: Project administration. Z.Y.: Project administration. L.H.: Formal analysis, funding acquisition. L.S.: Formal analysis, funding acquisition. S.H.: Conceptualization, formal analysis, funding acquisition, methodology, and writing—review and editing.

Funding

This work was supported by the National Science Foundation of China (82104053), Natural Science Foundation of Shandong Province (ZR2019BH035, ZR2020MD094, and ZR2021QC084), the project of youth creation team in Shandong higher education institutions (2022KJ26), and the Science and Technology Development Program in Weifang (2020GX014 and 2021GX053).

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

This study was carried out in accordance with the principles of the Basel Declaration. This study was approved by Experimental animal Ethics Committee, Weifang Medical College (2021SDL231).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.