INTRODUCTION:

Osteoporosis is a skeletal condition involving progressive bone loss and micro-architectural deterioration, leading to increased bone fragility and susceptibility to fractures. It is the most prevalent metabolic bone disease among adults worldwide¹. The prevalence of osteoporosis is expected to increase significantly in the future because of aging of the population. Osteoporosis mainly occurs in postmenopausal women and elderly men². In this current period of time osteoporosis is a serious health issue to deal with which is having a high prevalence worldwide.

Over 0.2 billion people suffer from osteoporosis, 30% of the postmenopausal women have osteoporosis in Europe and US. 15-30% of men and 40% women withstand one or more delicateness ruptures in their lifespan³. The pathophysiology of bone loss in osteoporosis is because of osteoclast and osteoblast imbalance^4,5. Estrogen deficiency, low calcium intake and high cholesterol also leads to osteoporosis⁶.

Alendronate is an anti-osteoporotic drug and first line therapy for osteoporosis. It falls under the oral bisphosphonates that are classified into non-nitrogen containing and nitrogen- containing (alendronate). Nitrogen containing bisphosphonates interfere with bone metabolite by inhibiting farnesyl disphosphate synthase in 3-hydroxy-3-methylglutaryl- CoA (HMG- CoA) reductase pathway. They stimulate the osteoclast apoptosis there by decrease osteoclast cells and bone resorption⁷. Depression frequently influence women all over their life time which results in reduced bone mineral density (BMD), even the post-menopausal women have an increased chance of developing depression in their life time where it decrease the quantity of bone mass⁸. Some clinical studies showed that anti-depressive agents may worsen the condition of osteoporosis⁹. Certain standard anti-depressive drugs such as serotonin and nor-adrenaline reuptake inhibitors (SNRIs), selective serotonin reuptake inhibitors (SSIRs), tri-cyclic anti-depressants (TCAs), there will be chance of having negative effects, they have been associated with increased chance of bone fractures¹⁰. The correlation between anti-depression and osteoporosis can be because of serotonin reuptake in brain, some animal studies have revealed that the treatment of anti-depressant shown the dysregulation in skeletal structure, diminution in bone mineral density and bone mechanical properties^11,12. The present study was aimed to comparative effects of venlafaxine and alendronate on biochemical, bone mechanical and anti-inflammatory properties in osteoporosis rats.

MATERIALS AND METHODS:

Animals:

Thirty six female Wistar rats weighing 220- 240g, were procured from the animal house, JSS College of Pharmacy, Ooty, India. The animal house was well ventilated and animal had 12± 1 h day and night schedule. The animals were housed in large spacious hygienic cages during the course of experiment period and room temperature was maintained at 22±2°C and relative humidity (60-70%). The animals were fed with standard laboratory diet and water ad libitum. The experimental protocol was conducted as per “The Committee for the Purpose of Control and Supervision of Experiments on Animals” (CPCSEA) guidelines, New Delhi, India. (Approval No.: JSSCP/OT/M Pharm/11/ 2018-2019).

Chemicals and drugs:

The chemical and drugs used included alendronate and venlafaxine (Cipla, India). Drugs were prepared freshly and dissolved in tap water administered daily for 28 days. All the other reagents, chemicals and kits used in the present were of analytical grade.

Experimental procedure:

Rats were anaesthetized by intraperitoneal injection of Ketamine (50mg/kg) and Xylazine (20mg/kg). Two incisions were made in costal margin on both sides of flank. Both the ovaries were exemplified, ligated, excised and then the abdomen was closed.Rats were divided into six groups of six animals each: Group I (control non-ovariectomized rats; NOVX): Rats of this group received 1ml distilled water orally for 28 days and served as a control group; Group II (Sham-operated group): Rats of this group were subjected to two horizontal incisions beneath the costal margin in the right and left flanks and the incision was closed without ovariectomy and left with no treatment and received 1 ml distilled water for 28 days. Group III (ovariectomized ‘OVX’, non-treated group): bilateral ovariectomy was done to rats of this group and 1 ml distilled water received for 28 days. Group IV (OVX, alendronate group): as in group III then treated with alendronate orally in a dose of 3mg/kg for 28 days; Group V: (OVX, venlafaxine-treated group): as in group III and then received venlafaxine orally in a dose of 20mg/kg for 28 days; Group VI: (OVX, alendronate-venlafaxine group): as in group III and then treated with alendronate and venlafaxine orally in a dose of 3 and 20mg/kg for 28 days respectively¹³. Body weight was taken in initial and final day. Serum ALP, calcium, and inflammatory cytokines were analyzed on 28^th day of the study. Animal were sacrificed bones (femur and tibia) were isolated. Immediately after isolation the bones (femur and tibia) were weighed. The three point bending test and bone mineral mass for femur were measured.

Biochemical estimation:

Blood was collected from retro-orbital puncture before the experiment and repeated every two weeks. After collection of blood samples were incubated at 37ºC until blood clotted. Separation of serum was done by centrifugation. Then the following parameters were measured in serum

Serum alkaline phosphatase:

Serum alkaline phosphate was estimated using alkaline phosphate kit (Q-line) by Auto-analyzer (Micro lab 200). In presence of Mg2+ and di-ethanolamine as phosphate acceptor, p-nitrophenylphosphatase is transformed by alkaline phosphatase into phosphate and p- nitrophenol (yellow compound). The reagent was prepared by reagent R1 800μl, reagent R2 200μl and serum 20μl was taken and incubated for 1 min at 25°C. Wavelength is set to 405nm readings were taken for all the groups. Calculated using following formula ALP (U/L) = △OD/ min Χ 2750^14,15.

Serum calcium:

Serum calcium was estimated using calcium kit (Ensure Biotec. Pvt. Limited) using Auto-analyzer. Aarsenazo III 1ml was taken as blank. Standard reading was taken using aarsenazo III 1ml and calcium standard 10μl. For test, serum 10μl and aarsenazo III 1ml was taken¹⁶. Calculated using following formula Serum calcium in mg/dl = Abs. of test/Abs. of Stdx10

Three point bone bending test:

Instrument was made up of wood similar to three point bending instruments. Bone was placed in the two triangle which serves as base 2 point and another point is present perpendicular to it which serves as third point. A constant force was applied to bone by rotating the screw present at top. Bone breaking time was noted manually for each group.

Initially right femur bones were weighed balance. Weight of the crucible dish was taken and bone was placed in crucible and weight was taken. Muffle furnace was set to the temperature of 600°C. The bone was placed in muffled furnace for 4 hours. The crucible was cooled to room temperature and weighed. The difference between before heating and after heating gives the weight of the ash content^17,18.

Estimation of inflammatory cytokines:

Serum levels of TNF-α, IL-1β, and IL-6 were determined using commercially available rat ELISA kits (R&D Systems, Minneapolis, USA)

Statistical analysis:

The data is represented as mean ± SEM. Body weight of the animal were analyzed by two way ANOVA while biochemical and mechanical properties were analyzed by one way ANOVA followed by Bonferroni multiple post test p values (<0.05) was considered notably. The analysis was carried using graph pad prism 6 software.

RESULTS:

Effects on drugs on body weight in OVX rats:

Rats of SHAM-operated group (group II) did not show any significant changes in body weight with NOVX rats (group I). The body weight has been significantly (p<0.05) increased in OVX group when compared to SHAM group. Alendronate treated rats showed significant (p<0.05) decrease while venflaxine treated rats showed significant increase (p<0.05) when compared to OVX induced rats. However, 28 days concurrent treatment with alendronate and venlafaxine showed significant (p<0.05) decrease in body weight when compared to OVX rats are shown in table 1.

Table 1: Effect of alendronate (3 mg/kg) and venlafaxine (20 mg/kg) on body weight

Group	Body weight(g)
Group	Initial	Final
NOVX	220.66±2.29	234.33±3.33
SHAM	222.16±2.77	236.66±4.41
OVX	223.83±3.20	256.83±2.66 ^a
OVX+ ALN	228.16±2.44	239.33±2.66 ^b
OVX + VEN	218.0±2.22	253.16±2.52^b,c
OVX+ ALN+ VEN	226.0±3.70	250.83±3.46^b,c

Results are expressed as mean ± SEM (n=6) NOVX: Non ovariectomized, OVX: Ovariectomized, ALN: Alendronate, VEN: Venlafaxine.

^a Significantly different from NOVX group (p < 0.05).

^b Significantly different from OVX group(p < 0.05).

^c Significantly different from OVX+ ALN group (p < 0.05).

Effects of drugs on serum ALP and calcium in OVX rats:

Rats of SHAM-operated group (group II) did not show any significant changes in serum ALP and calcium with NOVX rats (group I). The serum ALP level showed significant (p<0.05) increase in OVX group when compared to SHAM group. Venlafaxine, alendronate and concurrent administration of alendronate and venlafaxine significant (p<0.05) decrease when compared to OVX group. However concurrent administration of alendronate and venlafaxine treated group showed significant (p<0.05) increase when compared to alendronate treated rats are presented in table 2.

Table 2: Effect of alendronate (3 mg/kg) and venlafaxine (20 mg/kg) on serum ALP and calcium

Group	Serum ALP (U/L)	Serum calcium (mg/dl)
NOVX	64.17±8.47	8.50±0.79
SHAM	63± 8.9	9.00 ± 0.32
OVX	125 ±13.58^a	12.49 ± 0.23^a
OVX+ ALN	67.17± 9.08^b	8.55 ± 0.21^b
OVX + VEN	94.50 ±12.29^b	11.66 ± 0.20^b
OVX+ ALN+ VEN	87 ±7.07^b,c,d	10.35 ± 0.37^b,c,d

Results are expressed as mean±SEM (n=6)NOVX: Non ovariectomized, OVX: Ovariectomized, ALN: Alendronate, VEN: Venlafaxine.

^a Significantly different from NOVX group (p < 0.05).^b Significantly different from OVX group(p < 0.05).

^c Significantly different from OVX+ ALN group (p < 0.05).

^d Significantly different from OVX+ VEN group (p < 0.05).

Effects of drugs on bone mass in OVX rats:

Rats of SHAM-operated group (group II) did not show any significant changes in bone mass with NOVX rats (group I). The OVX induced rats showed significant (p<0.05) decrease in bone mass of femur and tibia when compared to SHAM group. Alendronate and concurrent administration of alendronate and venlafaxine significant (p<0.05) increased when compared to OVX group while venlafaxine did not show any significant changes respectively. However concurrent administration of alendronate and venlafaxine treated group showed significant (p<0.05) decrease when compared to alendronate treated rats are presented in table 3.

Table 3: Effect of alendronate (3 mg/kg) and venlafaxine (20 mg/kg) on bone mass

Group	Bone weight(g)
Group	Right femur	Right tibia
NOVX	1.04±0.03	0.99±0.06
SHAM	1.01 ± 0.06	0.98 ± 0.07
OVX	0.79 ± 0.8^a	0.71 ± 0.06^a
OVX+ ALN	0.97 ± 0.10^b	0.96 ± 0.07^b
OVX + VEN	0.77 ± 0.08	0.71 ± 0.06
OVX+ ALN+ VEN	0.92 ± 0.05^b,c,d	0.84 ± 0.07^b,c,d

Results are expressed as mean±SEM (n=6) NOVX: Non ovariectomized, OVX: Ovariectomized, ALN: Alendronate, VEN: Venlafaxine.

^a Significantly different from NOVX group (p < 0.05).

^b Significantly different from OVX group (p < 0.05).

^c Significantly different from OVX+ ALN group (p < 0.05).

^d Significantly different from OVX+ VEN group (p < 0.05).

Effects of drugs on TPB test in OVX rats:

Rats of SHAM-operated group (group II) did not show any significant changes in TPB with NOVX rats (group I). The result showed that there is a significant (p<0.05) decrease in time for TPB test in OVX group when compared to SHAM group. Alendronate and concurrent treatment of alendronate and venlafaxine significant (p<0.05) increase when compared to OVX group while venlafaxine did not show significant change respectively. However, concurrent administration of alendronate and venlafaxine showed significant (p<0.05) decrease when compared to alendronate group are presented in table 4.

Effects of drugs on bone mineral mass in OVX rats:

Rats of SHAM-operated group (group II) did not show any significant changes in bone mineral mass with NOVX rats (group I). The result showed that there is a significant (p<0.05) decrease in bone mineral mass in OVX group when compared to SHAM group. Alendronate and concurrent treatment of alendronate and venlafaxine significant (p<0.05) increase when compared to OVX group while venlafaxine did not show significant change respectively. However, concurrent administration of alendronate and venlafaxine showed significant (p<0.05) decrease when compared to alendronate group are presented in table 4.

Table 4: Effect of alendronate and venlafaxine on bone mineral mass and TPB bending test

Group	Bone mineral mass Ash weight (mg)	TPB bending test Time (min)
NOVX	0.25 ± 0.02	6.65±0.24
SHAM	0.23 ± 0.04	6.68±0.37
OVX	0.16 ± 0.03^a	3.67±0.47^a
OVX+ ALN	0.23 ± 0.06^b	6.04±0.57^b
OVX + VEN	0.17 ± 0.05	3.57±0.45
OVX+ ALN+ VEN	0.21 ± 0.06^b,c,d	4.57±0.50^b,c,d

Results are expressed as mean±SEM (n=6) NOVX: Non ovariectomized, OVX: Ovariectomized, ALN: Alendronate, VEN: Venlafaxine.

^a Significantly different from NOVX group (p < 0.05).

^b Significantly different from OVX group (p < 0.05).

^c Significantly different from OVX+ ALN group (p < 0.05).

^d Significantly different from OVX+ VEN group (p < 0.05).

Effects of drugs on inflammatory cytokines in OVX rats:

Effect of alendronate and venlafaxine on the concentration of serum inflammatory markers in sham-operated control and experimental rats were presented in Table.5. The concentrations of inflammatory markers like TNF-α, IL-1β, and IL-6 of ovariectomized rats were significantly increased (p < 0.05) compared with sham-operated rats. Alendronate and concurrent treatment of alendronate and venlafaxine significant (p<0.05) decrease when compared to OVX group while venlafaxine did not show significant change respectively. However, concurrent administration of alendronate and venlafaxine showed significant (p<0.05) increase when compared to alendronate group are presented in table 5.

Table 5: Effect of alendronate (3 mg/kg) and venlafaxine (20 mg/kg) on concentration of serum inflammatory markers

Group	TNF-α (ng/mL)	IL-1β (pg/mL)	IL-6 (pg/mL)
NOVX	11.20±0.03	65.80±0.16	94.20±0.15
SHAM	11.39±0.02	67.79±0.17	95.74±0.25
OVX	20.52±0.50^a	96.15±0.34^a	167.23±0.33^a
OVX+ ALN	14.07±0.31^b	72.90±0.18^b	109.12±0.26^b
OVX + VEN	18.69±0.36	68.06±0.273	167.16±0.20
OVX+ ALN+ VEN	16.06±0.21^b,c,d	84.62±0.23^b,c,d	130.3±0.37^b,c,d

Results are expressed as mean±SEM (n=6) NOVX: Non ovariectomized, OVX: Ovariectomized, ALN: Alendronate, VEN: Venlafaxine.

^a Significantly different from NOVX group (p < 0.05).

^b Significantly different from OVX group (p < 0.05).

^c Significantly different from OVX+ ALN group (p < 0.05).

^d Significantly different from OVX+ VEN group (p < 0.05).

DISCUSSION:

Postmenopausal osteoporosis is prevalent condition usually associated with aging and decline in ovarian function. The main clinical manifestation of this metabolic disorder is fragility fracture because of imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation^19,20. Venlafaxine is a known and clinically used anti- depressant drug which worsens the condition of osteoporosis. Serotonin released from the brain through endogeneous neurotransmitters (5HT2c) receptor on the neurons of ventromedial hypothalamic (VMH) reduces sympathetic activity leads to decrease in bone formation. This drug blocks the serotonin reuptake and will worsen the condition of osteoporosis^21-24. Our study revealed that venlafaxine treated rats does not show anti- osteoporotic activity. Marked increase in body weight of the OVX group uses the success of ovariectomy in rats, because estrogen deficiency directly increases the body weight. In this study, Alendronate showed significant (p<0.05) difference in body weight when compared to OVX group. Concurrent administration of alendronate and venlafaxine has decrease in body weight when compared to alendronate.

In the present study, osteoporosis was induced by bilateral ovariectomy resulted in significant increase in the serum level of serum ALP and serum calcium level; These results matched those of Yoon and co-workers who detected significant elevation of serum ALP and calcium in ovariectomized rats²⁵. Concurrent administration of alendronate and venlafaxine showed increase in serum calcium and ALP. Therefore, bone resorption and formation markers increase as a compensatory mechanism to fill the higher number of resorption cavities. Calcium in serum indicates bone remodeling. ALP as one of the bone formation markers is found in the membrane of osteoblasts and released into the circulation during bone formation²⁶. The ovariectomy in rat decreases the mineral content in the bones which was observed in the study. The ovariectomized group showed a decrease in the ash values. Treatment of the ovariectomized group with the alendronate prevented the reduction ash values showing its effectiveness in the prevention of bone loss. Concurrent administration of alendronate and venlafaxine shows decreased in ash value when compared to alendronate treated rats. Due to the small stimulatory effect of growth hormone on longitudinal growth through its effect on the pituitary gland^27,28. The weights of the bones were increased in alendronate when compared to OVX induced rats.

Concurrent administration of alendronate and venlafaxine shows decrease in a bone weight when compared to alendronate treated rats. There was no significant increase/decrease in the length and diameter in all the rats. Three point bending test demonstrated the worsening of mechanical property of femur bone in OVX rats. Concurrent administration of alendronate and venlafaxine has decrease in activity when compared to alendronate treated rats.

Deficiency of estrogen in OVX mice can trigger free radical generation in bone marrow and subsequently initiate the inflammatory cascade by up regulating the expression of various pro-inflammatory cytokines particularly TNF-α through activating T cells²⁹. Our study also revealed the concentrations of inflammatory markers like TNF-α, IL-1β, and IL-6 of ovariectomized rats were significantly increased (p < 0.05) compared with sham-operated rats. On comparison with OVX group, ALN treated group and concurrent administration of ALN and VEN markedly lowered the concentrations of those inflammatory markers. Our results also supported and inferred that the serum levels of IL-1β, IL-6 and TNF-α were concomitantly increased in OVX induced rats as compared to sham-operated rats^30,31.

CONCLUSION:

In conclusion, venlafaxine is a serotonin and epinephrine reuptake inhibitor class of anti- depressant drug which inhibit brain serotonin which leads to inhibit bone formation. The concurrent administration of alendronate and venlafaxine in OVX induced rats showed decrease in serum ALP and calcium, body weight, bone weight and bone mineral mass when compared to alendronate. Hence, from the above findings the combination of alendronate and venlafaxine showed worsen the condition osteoporosis. Further studies can be carried out to understand the exact mechanism of concurrent administration of both the drugs in treatment of osteoporosis and depression.

ACKNOWLEDGEMENT:

The authors would like to thank DST-FIST, New Delhi for their infrastructure support to our Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, The Nilgiris, Tamilnadu.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest to disclose.

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Received on 28.07.2020 Modified on 26.09.2020

Research J. Pharm. and Tech. 2021; 14(7):3553-3558.

DOI: 10.52711/0974-360X.2021.00615