Inflammopharmacology

https://doi.org/10.1007/s10787-021-00849-0 Inflammopharmacology
ORIGINAL ARTICLE

Venlafaxine demonstrated anti‑arthritic activity possibly

through down regulation of TNF‑α, IL‑6, IL‑1β, and COX‑2
Mater Hussen Mahnashi1 · Zeeshan Jabbar2 · Alamgeer3   · Hafiz Muhammad Irfan2 · Mulazim Hussain Asim2 ·
Muhammad Akram2 · Ahmed Saif4 · Mohammed Abdulrahman Alshahrani4 · Mohammed Ali Alshehri4 ·
Saeed Ahmed Asiri4

Received: 10 May 2021 / Accepted: 7 July 2021

© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021

Abstract
Venlafaxine is a serotonin-norepinephrine reuptake inhibitor used to treat depression. Previous studies demonstrated its anti-
nociceptive and anti-inflammatory activities through the suppression of pro-inflammatory cytokines. Present research aimed
to explore its anti-arthritic potential. Different in-vitro assays including egg albumin, bovine serum albumin denaturation
and human red blood cell (RBC) membrane stabilization assays along with in-vivo models of formaldehyde and complete
Freund’s adjuvant-induced arthritis were used to study its anti-arthritic effect. Venlafaxine inhibited egg albumin and bovine
serum albumin denaturation and preserve the integrity of red blood cells membrane in concentration-dependent manner.
In formaldehyde-induced arthritis venlafaxine significantly (p < 0.001) reduced the paw edema on treatment for 10 days.
Chronic administration of venlafaxine for 28 days in Freund’s adjuvant-induced arthritis model decreased the paw volume
(p < 0.001), arthritic index (p < 0.01), flexion pain score (p < 0.05), mobility score (p < 0.05), and improved the stance score
(p < 0.05). Venlafaxine also significantly declined the rheumatoid factor (p < 0.01) and C-reactive protein (p < 0.05) levels
and increased the RBC count (p < 0.01) and Hb value (p < 0.001). Upon PCR analysis venlafaxine remarkably turndown the
mRNA expression of TNF-α, IL-6, IL-1β, and COX-2. Taken together it is inferred from current findings that venlafaxine
possesses the significant anti-arthritic activity and could be a potential therapeutic option for the treatment of rheumatoid
arthritis.

Keywords  Venlafaxine · Arthritis · Bovine serum albumin · Freund’s adjuvant · PCR

Introduction and destruction of joint tissues that restrict its movement

Rheumatoid arthritis (RA) is a chronic disease of joints
that results from over activation of the immune system. It
is characterized by pain, morning stiffness, inflammation,
* Alamgeer
alam_yuchi@yahoo.com
1
Department of Pharmaceutical Chemistry, College
of Pharmacy, Najran University, Najran 61441,
Kingdom of Saudi Arabia
2
College of Pharmacy, Faculty of Pharmacy, University
of Sargodha, Sargodha 40100, Pakistan
3
Punjab University College of Pharmacy University
of the Punjab, Lahore 54000, Pakistan
4
Department of Clinical Laboratory Sciences, Faculty
of Applied Medical Sciences, Najran University, 1988,
Najran 61441, Saudi Arabia
(Choudhary et al. 2014). Both small and large types of joints
may be involved in RA, but distal interphalangeal joints
are affected rarely (Aletaha and Smolen 2018). In addi-
tion, arthritic condition may be accompanied by a variety
of extra-articular symptoms including rheumatoid nodules,
vasculitis, pulmonary involvement, and systemic comorbidi-
ties that seriously affect patient health (Smolen et al. 2016;
Yu et al. 2018). The worldwide occurrence of rheumatoid
arthritis is approximately 0.5–1%. In 2014, the pervasiveness
of RA ranged from 0.53 to 0.55% in the American popula-
tion, affecting about 1.28–1.36 million people (Hunter et al.
2017). Women are 2–3 times more prone to develop rheuma-
toid arthritis as compare to men (Ngo et al. 2014).
Development of RA is initiated when genetic and environ-
mental factors work on predisposing genotype, which results
in inflammation and deleterious synovial response (Makry-
giannakis et al. 2008). Antigenic exposure to the genetically

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predisposed individuals (HLA-DR), causes CD4 + T-Cell acti-
vation. These cells secrete cytokines such as TNF-α, inter-
leukin IL-1, and IL-6 that further activate endothelial cells,
macrophages, and B lymphocytes. All these activated cells
cause damage to joint tissue and pannus formation (Shilpa
et al. 2018).
The primary objectives in the management of RA are reduc-
tion in pain and inflammation, joint function preservation, and
prevention of joint deformity. Pharmacological management
of RA involves the use of disease-modifying antirheumatic
drugs (DMARDs), glucocorticoids, and non-steroidal anti-
inflammatory drugs (NSAIDs) (Alamgeer et al. 2015). The
long-term use of these drugs has several adverse effects includ-
ing neutropenia, thrombocytopenia, osteoporosis, hepatotoxic-
ity, immunodeficiency, and gastric irritation (Papadakis et al.
2019). Other limitations are there is a high cost of therapy
and therapeutic outcomes are not fully achieved. Therefore,
the discovery of new drugs for a batter cure of rheumatoid
arthritis is needed.
Venlafaxine structurally belongs to phenylethylamine, is a
known anti-depressant drug. Its anti-depressant effect is pro-
duced by serotonin and norepinephrine reuptake inhibition
hence increased neurotransmitters concentration in synaptic
cleft. The pain-relieving properties of venlafaxine has been
demonstrated in different pain models such as fibromyalgia,
neurogenic pain, and diabetic neuropathy (Cegielska-Perun
et al. 2013; Dwight et al. 1998; Sumpton and Moulin 2001).
Recently, anti-inflammatory and anti-nociceptive effects of
venlafaxine were studied in various models. It significantly
reduces inflammation and pain in carrageenan-instigated paw
edema in rats and decreases myeloperoxidase (MPO) activity,
IL‐1β and TNF‐α production (Aricioğlu et al. 2005; Chugh
et al. 2013; Ganesh et al. 2017; Hajhashemi et al. 2015). Ven-
lafaxine improves inflammatory injury in ulcerative colitis
induced in normal and depressed rats by acetic acid. This effect
is supposed to be mediated by its anti-depressant and anti-
inflammatory activity (Minaiyan et al. 2015). It ameliorates
autoimmune encephalomyelitis by inhibiting expression of
TNF-α, IFN-γ and IL-12 p40 that depicts its immunomodula-
tory activity (Vollmar et al. 2009). In astrocytes and micro-
glia cell culture model venlafaxine exhibits anti-inflammatory
effect through decreasing pro-inflammatory (IFN-γ and IL-6)
cytokines and increasing TGF-β release (Vollmar et al. 2008).
Keeping in view of its anti-nociceptive and anti-inflamma-
tory potential, the present study was designed to explore the
(Absorbance of Control − Absorbance of Sample)
Percentage Inhibition =
Absorbance of Control
13
M. H. Mahnashi et al.
anti-arthritic activity of venlafaxine through several in-vitro
and in-vivo models.
Material and methods
Drugs, chemicals, and regents
Venlafaxine (Standpharm, Lahore), Piroxicam (Sigma-
Aldrich), phosphate buffer (pH 6.4, pH 6.3), phosphate buffer
(0.15 M, pH 7.4), bovine serum albumin (Sigma-Aldrich),
HCl, Alsver’s solution, formaldehyde, Complete Freund’s
adjuvant (Sigma-Aldrich), chloroform, formalin, isopro-
panol, ethanol, TriZol reagent (Sigma-Aldrich), ­WizScript™
cDNA Synthesis kit (Wizbiosolutions), and amaR OnePCR
(GeneDirex).
Animals and housing conditions
For in-vivo inflammatory models, Sprague–Dawley rats
of both gender (male and female) having a weight range of
150–250 g were used. All animals were housed under a 12-h
light/dark cycle at 25 ± 2 °C with a humidity level of 55 ± 5%.
There was free access to food and water to animals and 1 week
acclimatization period was given before the start of experi-
ments. All the protocols involving animals were approved by
the Institutional Animal Ethical Committee, College of Phar-
macy, University of Sargodha, Pakistan with approval number
UOS/112/18 dated 12/20/18.
In‑vitro assays for anti‑arthritic property
Assessment of anti‑arthritic activity of venlafaxine by egg
albumin denaturation assay
For assessment of egg albumin denaturation prevention abil-
ity of venlafaxine, 0.2 mL fresh egg albumin, 2 mL of each
drug concentration (50–6400 µg/mL), and 2.8 mL phosphate
buffer of pH 6.4 were put together in a test tube to make total
5 mL of the reaction mixture. The control solution was com-
prised of egg albumin, phosphate buffer, and distilled water.
The standard drug used in this assay was piroxicam. Prepared
test tubes having reaction mixture were put for incubation of
15 min at 37 ± 2 °C, later on, reaction mixtures were heated
at 70 ± 2 °C for 5 min. After cooling of samples absorbance
of each sample was measured at 660 nm using a UV–Visible
spectrophotometer then percentage inhibition was calculated
by following formula (Uttra and Alamgeer 2017).
× 100
Venlafaxine demonstrated anti‑arthritic activity possibly through down regulation of TNF‑α,…
Assessment of anti‑arthritic activity of venlafaxine
by bovine serum albumin (BSA) denaturation assay
Bovine serum albumin (BSA) denaturation assay is another
heat-induced protein denaturation test to study the anti-
arthritic property of test compounds. This test was carried
out by preparing series of solutions in test tubes, for instance,
test solutions, product control solution, and test control solu-
tion. Test solutions include 0.45 mL BSA (5% w/v solution
in distill water) and 0.05 mL of various drug concentrations
(50–6400 µg/mL). Test control solution compromise 0.45 mL
of BSA and 0.05 mL distill water, whereas product control
solutions consist of 0.45 mL distilled water and 0.05 mL drug
concentrations.
Piroxicam was used as a standard drug. Each solution was
adjusted to a pH of 6.3 using 1 N HCl. Then all solutions were
put at 37 ± 2 °C for 20 min incubation after that temperature
was raised to 57 ± 2 °C for half an hour. Later on, solutions
were cooled and 2.5 mL of phosphate buffer (pH 6.3) was
added to each solution, and turbidity was estimated at 660 nm
(Alamgeer et al. 2017b). The following formula was used to
calculate the percentage inhibition of protein denaturation:
Percentage inhibition = 100 − (Abs Ts − Abs Pc)∕ Abs Tc × 100
where Abs shows absorbance, Ts is test solution, Pc reflects
product control solution and Tc = represents test control
solution.
Assessment of anti‑arthritic activity of venlafaxine using
human red blood cell (HRBC) membrane stabilization assay
To prepare HRBC suspension, a blood sample was obtained
from a healthy person who had not administered NSAID for
14 days before the time of sample. Collected blood was mixed
with the same volume of Alsver’s solution and centrifuged at
3000 rpm for 20 min. Packed cells were removed and washed
three times with isosaline solution (0.85%), then 10% v/v
HRBC suspension was prepared in isosaline solution. Sample
mixture was prepared by adding 1 mL different drug concen-
trations (50–6400 µg/mL), 1 mL phosphate buffer (0.15 M, pH
7.4), 2 mL hyposaline solution (0.36%), and 0.5 mL of HRBC
suspension (10% v/v). The control mixture was comprised of
phosphate buffer, HRBC suspension, and 2 mL distilled water.
Samples were prepared in triplicate. Piroxicam was used as a
standard drug. All samples were incubated at 37 ± 2 °C for 30
min followed by centrifugation at 3000 rpm for 20 min. After
that absorbance of the supernatant was recorded at 560 nm
(Uttra and Alamgeer 2017). Percentage membrane stabiliza-
tion was estimated by formula:
Percentage of membrane stabilization = 100 − (Abs. of sample ∕ Abs. of control) × 100
In‑vivo models for anti‑arthritic study
Evaluation of anti‑arthritic activity
against formaldehyde‑induced arthritis
Anti-arthritic effect of venlafaxine was evaluated in the
acute model of arthritis (formaldehyde-induced arthri-
tis). Experimental rats were divided into five groups hav-
ing five animals in each group as follow: Group I shows
arthritic control (normal saline 10 mL/Kg), Group II rep-
resents venlafaxine 25 mg/Kg, Group III shows venlafax-
ine 50 mg/Kg, Group IV reflects venlafaxine 100 mg/Kg
and Group V represents piroxicam 10 mg/Kg.
Respective treatments were administered orally for
10  days. Arthritis was induced by 0.1  mL subplanter
injection of formaldehyde (2% solution) into left hind paw
30 min after oral dose at day 1 and day 3. Paw volume was
determined on the 2nd, 4th, 6th, 8th, and 10th day using
a digital plethysmometer (Uttra and Alamgeer 2017). The
percentage of paw volume inhibition was computed by
the formula:
Vc − Vt
Percentage inhibition = × 100
Vc
where Vc is paw volume of control, and Vt is paw volume
of treated sample.
Evaluation of anti‑arthritic activity against Complete
Freund’s Adjuvant‑induced arthritis
Long-term anti-arthritic activity of venlafaxine was eval-
uated in Complete Freund’s Adjuvant-induced arthritis
model. Arthritis was prompted by an already established
method with slight modifications (Ekambaram et al. 2010).
Animals were divided into four groups having six animals
in every group as follow: Group I is normal control (nor-
mal saline 10 mL/Kg), Group II is arthritic control (nor-
mal saline 10 ml/Kg), Group III is venlafaxine 100 mg/
Kg and Group IV is piroxicam 10 mg/Kg.
The length of the model was 28 days and all animals
received respective treatment during the whole study
period. On day 1, half an hour later than oral drug admin-
istration arthritis was induced by injection of Complete
Freund’s Adjuvant (0.1 mL) in subplanter region of the left
hind paw of animals except Group I. Parameters like body
weight, arthritic index, and paw volume were assessed
on 0, 7, 14, 21 and 28  days. Arthritic index was com-
puted according to criteria used by Gohil et al. in which
13

ears, nose, tail, forepaws, and hind paws of each animal
were observed and scored as ears (0 = absence of nodules,
1 = presence of nodules), nose (0 = no swelling of connec-
tive tissue, 1 = intensive swelling of connective tissue),
tail (0 = absence of nodules, 1 = presence of nodules),
forepaws (0 = Absence of inflammation, 1 = Inflammation
of at least one joint), hind paws (0 = absence of inflamma-
tion, 1 = slight inflammation, 2 = moderate inflammation,
3 = marked inflammation) (Gohil et al. 2018). Flexion pain
score, mobility score, and stance score were determined on
14 and 28 days by prescribed criteria employed by (Patil
et al. 2011). On, 28th day animals were anesthetized with
chloroform and blood samples were collected by cardiac
puncture. For radiographic analysis, arthritic legs of ani-
mals were amputated from the knee joint, after that pre-
served in 10% formalin. Histopathology was carried out
by making 5-mm thick sections of ankle joint and stained
with hematoxylin–eosin (H and E) later observed by light
microscope. Hematologic parameters were analyzed on
28th day from blood samples. Blood biomarkers includ-
ing ALT, AST, CRP, and RF level were also measured.
Furthermore, assessment of cellular mechanism mRNA
expression of different inflammatory mediators including
IL-6, TNF-α, IL-1β, and Cox-2 was determined by con-
ventional PCR.
Determination of TNF‑α, IL‑6, IL‑1β, and COX‑2
mRNA expression level
RNA isolation and reverse transcription
Total RNA was extracted from blood samples by Trizol
method. To synthesize cDNA by reverse transcription, pro-
tocol given by kit manufacturer ­(WizScript™ cDNA Syn-
thesis kit, Wizbiosolutions) was followed. Briefly, 500 ng
RNA template was mixed with 1 µL of dNTP mix, 1 µL of
oligo dT 20, and RNase free water to make a final volume of
10 µL. Then the mixture was heated at 65 °C for 5 minutes in
the thermal cycler then immediately cooled on ice. After that
10X reaction buffer (2 µL), RNase inhibitor (0.5 µL), RTase
(1 µL), DTT (1 µL), and RNase free water (5.5 µL) were
added into the previous mixture. Later on, reaction mixture
was incubated at 42 °C for 45 min and then heated at 70 °C
for 10 min to stop the reaction. The synthesized cDNA was
stored at – 20 °C.
Polymerase chain reaction (PCR) and gel electrophoresis
To carry out polymerase chain reaction, 1 µL of cDNA
template, 9 µL of PCR reaction mixture (amaR OnePCR,
GeneDirex), 1.5 µL of forward primer, 1.5 µL of reverse
primer, and 3 µL nuclease free water were mixed. The mix-
ture was put into thermal cycler having a setting of 35 cycles
13
M. H. Mahnashi et al.
of denaturation (95 °C for 30 s), annealing (58–62.3 °C for
30 s), and extension (72 ° C for 60 s). Finally, reaction was
stopped by heating 72 °C for 10 min. Then 6 µL of PCR
product was loaded on 1% agarose gel and visualized by gel
electrophoresis. ImageJ software (NIH) was used to semi-
quantify band intensity of PCR product by densitometry.
Primers of TNF-α, IL-6, IL-1β, COX-2, and GAPDH were
picked from previous study (Hasan et al. 2018) and synthe-
sized from commercial manufacturer. GAPDH was used as
reference gene.
Statistical analysis
Results were illustrated as mean ± SEM. Statistical signifi-
cance was determined by subjecting the data to different
tests, for all normally distributed data one-way ANOVA
followed by Dunnet’s test and two-way ANOVA followed
by Bonferroni post hoc test using Graph Pad prism. Effects
were considered significant at p < 0.05.
Results
Venlafaxine inhibited egg albumin denaturation
In the current research study, protein denaturation assay
was carried out by heating fresh egg albumin with different
concentrations of drugs. Venlafaxine inhibited denaturation
of egg albumin in concentration-dependent way. Maximum
inhibition of venlafaxine 66.97 ± 0.72% was recorded at a
concentration of 6400 µg/mL as shown in Fig. 1.
Venlafaxine inhibited bovine serum albumin (BSA)
denaturation
The present study showed that venlafaxine prevented protein
denaturation in BSA denaturation assay. Protein denatura-
tion inhibition was found in concentration-dependent pattern
ranging from 53.61 ± 0.70% to 78.81 ± 0.24% for venlafaxine
as reflected in Fig. 1.
Venlafaxine stabilized the HRBC membrane
Venlafaxine possesses membrane stabilization property as
shown in HRBC membrane stabilization assay. The ability
of membrane stabilization increased as the concentration of
drug raised. Maximum percentage membrane stabilization
of piroxicam and venlafaxine were found 48.64 ± 0.39%, and
50.00 ± 1.33% respectively at a concentration of 6400 µg/mL
as expressed in Fig. 1.
Venlafaxine demonstrated anti‑arthritic activity possibly through down regulation of TNF‑α,…

Fig. 1  Effect of venlafaxine on
protein denaturation assays and
HRBC membrane stabilization
assay. Results are expressed as
Mean ± SEM

Venlafaxine reduced the paw volume
in formaldehyde‑induced arthritis
Venlafaxine decreased rats paw volume compared to
arthritic control during the study period. The results
were found more significant on the 10th day. Ven-
lafaxine 100  mg/Kg showed maximum paw volume
reduction at end of study p < 0.001 is represented in
Table 1.

13
 M. H. Mahnashi et al.

Table 1  Effect of venlafaxine on paw volume in formaldehyde induced arthritis

Experimental group Paw volume in mL (% reduction)
Day 2 Day 4 Day 6 Day 8 Day 10

Arthritic Control 1.584 ± 0.020 1.732 ± 0.022 1.656 ± 0.021 1.626 ± 0.028 1.586 ± 0.022

Venlafaxine 25 mg/kg 1.284 ± 0.070** 1.392 ± 0.102** 1.278 ± 0.102*** 1.244 ± 0.099*** 1.196 ± 0.094***
(18.94%) (19.63%) (22.83%) (23.49%) (24.59%)
Venlafaxine 50 mg/kg 1.428 ± 0.114 1.522 ± 0.092 1.406 ± 0.134* 1.326 ± 0.122** 1.182 ± 0.083***
(9.85%) (12.12%) (15.10%) (18.45%) (25.47%)
Venlafaxine 100 mg/kg 1.436 ± 0.070 1.574 ± 0.076 1.316 ± 0.062** 1.16 ± 0.038*** 0.866 ± 0.012***
(9.34%) (9.12%) (20.53%) (28.66%) (45.40%)
Piroxicam 10 mg/Kg 0.66 ± 0.042*** 0.916 ± 0.057*** 0.788 ± 0.046*** 0.706 ± 0.030*** 0.6 ± 0.031***
(58.33%) (47.11%) (52.42%) (56.58%) (62.17%)

Results are expressed as Mean ± SEM (n = 5), *p < 0.05 **p < 0.01, ***p < 0.001 compared to arthritic control analyzed by two-way ANOVA
followed by Bonferroni’s multiple comparison test

Venlafaxine relieved the Complete Freund’s
adjuvant (CFA)‑induced arthritis
In CFA-induced arthritis model arthritic control rats had
a significant increase in paw volume compare to normal
control (p < 0.001). Venlafaxine-treated animals paw had
decreased volume on the 7th, 14th, 21th, and 28th days.
Volume reduction property of venlafaxine raised as the
number of days increased with maximum reduction at 28th
day p < 0.001 as exhibited in Fig. 2.
Arthritic index was scored by noting inflammation and
nodules on rat’s ear, nose, tail, and paws. Treatment of rats
with venlafaxine reduced the arthritic index in contrast to
arthritic rats but result was more significant (p < 0.01) on
28th day Fig. 3.
In the present study rats in arthritic control group had
significant weight reduction during the study period. At
first, venlafaxine-treated animals did not show weight loss
prevention while slight weight restoration was observed on
28 day of study. Piroxicam did not affect weight restoration
during the study period but weight loss was minimum com-
pared to arthritic control as shown in Table 2.
Arthritic control rats had elevated flexion pain during
the study period. Venlafaxine significantly attenuated the
pain in animals (p < 0.05). Piroxicam expressed maximum
pain reduction property (p < 0.001) at end of the study as
observed in Fig. 4a. Disturbance in the mobility of arthritic
control rats was observed in the current study and venlafax-
ine treatment for 28 days significantly reduced the mobility
score (p < 0.05) and increased the stance score (p < 0.05)
in contrast to arthritic control. Similar effects were also

Fig. 2  Effect of venlafaxine on paw volume in CFA-induced arthritis. pared to arthritic control analyzed by two-way ANOVA followed by
Results are expressed as Mean ± SEM (n = 6), # # #p < 0.001 com- Bonferroni’s multiple comparison test
pared to normal control and *p < 0.05 **p < 0.01, ***p < 0.001 com-

13
Venlafaxine demonstrated anti‑arthritic activity possibly through down regulation of TNF‑α,…

Fig. 3  Effect of venlafaxine on arthritic index in CFA-induced arthritis. Values are expressed as Mean ± SEM (n = 6), **p < 0.01 compared with
arthritic control by two-way ANOVA followed by Bonferroni’s multiple comparison test

Table 2  Effect of venlafaxine on the weight of animals in CFA induced arthritis

Group Weight (g) at Weight (g) at Weight (g) at Weight (g) at Weight (g) at
Day 0 Day 7 Day 14 Day 21 Day 28

Normal control 189.83 ± 9.52 201 ± 7.14 206.5 ± 9.75** 216.83 ± 11.71*** 217.83 ± 12.73***

Arthritic control 205.67 ± 10.27 180 ± 7.75 172 ± 7.10 169.50 ± 6.23 151.67 ± 6.85
Venlafaxine 100 mg/Kg 189 ± 6.28 168.33 ± 6.09 164.33 ± 6.52 160.83 ± 6.36 170.67 ± 7.53
Piroxicam 10 mg/Kg 190.50 ± 2.85 181.50 ± 2.77 177.83 ± 3.32 174 ± 4.47 166.50 ± 3.69

Values are expressed as Mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001 compared with arthritic control by two-way ANOVA followed
by Bonferroni’s multiple comparison test

observed by standard drug piroxicam as expressed in Fig. 4b
and c.
Administration of CFA injection in rats caused a fall in
RBC and Hb level that indicate the presence of anemia.
Venlafaxine and standard drug piroxicam significantly
(p < 0.01 and p < 0.001) improved RBC count and Hb con-
tent as shown in Table 3. Besides this, the level of total leu-
kocyte, platelets, ALT, AST, ESR, CRP and RF values were
observed elevated in arthritic control rats. Oral administra-
tion of venlafaxine and piroxicam (p < 0.05–0.001) reduced
the level of these parameters as shown in Table 3.
During radiographic analysis joint swelling, joint space
narrowing, bone erosion and joint deformities in arthritic
control rats were observed. Venlafaxine and standard
drug halted these changes as given in Fig. 5. Histopatho-
logical examination of paw revealed structural damage and
increased immune cells infiltration at the site of inflamma-
tion in the arthritic control group. Venlafaxine treatment
decreased the structural damage and cells infiltration as
illustrated in Fig. 6.
Conventional PCR of blood samples indicated an ele-
vation of mRNA expression of TNF-α, IL-1β, IL-6, and
COX-2 in arthritic control rats. Oral administration of ven-
lafaxine for 28 days significantly reduced TNF-α expression
(p < 0.05) as shown in Fig. 7a. A notable decrease in mRNA
level of IL-1β was also seen in venlafaxine (p < 0.05) treated
group Fig. 7b. Further, venlafaxine showed declined IL-6
and COX-2 mRNA level (p < 0.05) Fig. 7c, d.
Discussion
The objective of the current research work was to evaluate
the anti-arthritic activity of venlafaxine by utilizing in vitro
methods and in vivo animal models. Further, molecular
effects of venlafaxine on pro-inflammatory cytokines were
studied. Findings of the present study depicted arthritis
relieving property of venlafaxine by inhibition of protein
denaturation, stabilization of HRBC membrane, paw vol-
ume reduction and improving other parameters of arthritis
through down regulation of pro-inflammatory cytokines.
Protein denaturation is a process in which primary, sec-
ondary, tertiary and quaternary structure of protein is dis-
turbed due to heat, extrinsic stress, acid, base and organic

13
 M. H. Mahnashi et al.

Fig. 4  Effect of venlafaxine
on a flexion pain, b mobility, c
a
stance. Values are expressed as
Mean ± SEM (n = 6), *p < 0.05,
**p < 0.01, ***p < 0.001 com-
pared with arthritic control by
two-way ANOVA followed by
Bonferroni’s multiple compari-
son test

Table 3  Effect of venlafaxine Hematological parameters Arthritic control Normal control Venlafaxine 100 mg Piroxicam 10 mg
on hematological parameters in
CFA-induced arthritis 6
RBC × ­10 /µL 5.35 ± 0.13 9.06 ± 0.22*** 6.65 ± 0.31** 6.93 ± 0.17***
Hb g/dL 8.80 ± 0.49 14.87 ± 0.11*** 11.17 ± 0.26*** 11.6 ± 0.24***
TLC × ­103/µL 10.13 ± 0.60 2.80 ± 0.27*** 6.57 ± 0.97** 5.07 ± 0.84***
Platelets × 103/µL 920.20 ± 32.18 712.80 ± 50.41* 689 ± 53.04** 679.3 ± 51.65**
ALT U/L 142.50 ± 7.13 120 ± 4.03* 122.50 ± 5.06* 124.20 ± 4.4
AST U/L 330.80 ± 18.31 283.70 ± 13.11* 319.30 ± 8.86 308.70 ± 10.49
ESR mm/h 7.17 ± 0.70 3.15 ± 0.31*** 4.17 ± 0.70** 4.67 ± 0.49*
CRP mg/L 23.50 ± 3.06 4.33 ± 0.42*** 15.33 ± 1.50* 11.83 ± 1.80**
RF IU/L 17.17 ± 2.80 5.50 ± 0.76*** 8.67 ± 1.05** 9.33 ± 1.63*

Results are expressed as Mean ± SEM (n = 6) *p < 0.05, **p < 0.01, ***p < 0.001 compared with arthritic
control analyzed by one-way ANOVA followed by Dunnett’s test

13
Venlafaxine demonstrated anti‑arthritic activity possibly through down regulation of TNF‑α,…

Fig. 5  Representative radio-
graphs of paw showing the a b
extent of joint deformities
(arrow sign) in normal control
(a), arthritic control (b), venla-
faxine (c), and piroxicam (d)

c d

Fig. 6  Effect of venlafaxine
on histopathological changes
(inflammatory cells infiltration
and tissue damage) in ankle
joints of CFA-induced arthritic
rats

solvent. Changes in hydrogen, hydrophobic, electrostatic
and disufide bonding are the key mechanisms in protein
denaturation (Alamgeer et al. 2017b). Auto antigens pro-
duced by in vivo denaturation of protein in certain rheumatic
ailments causes activation of immune system that further
promote inflammation (Iffath and Caroline 2018). Many
anti-inflammatory drugs are reported having anti-protein
denaturation property including indomethacin, diclofenac
sodium, flufenamic acid, and salicylic acid (Alamgeer et al.
2015; Henneh et al. 2018). Therefore, chemicals having
protein denaturation inhibitory potential are the prospective
candidates for anti-inflammatory and anti-arthritic drug dis-
covery. In this study protein denaturation assays using egg
albumin and BSA were performed and venlafaxine exhibited
encouraging protein denaturation inhibition (Fig. 1). These
results showed that venlafaxine possesses anti-inflammatory
and anti-arthritic potential.
Lysosomes are vesicles found in inflammatory cells and
contain enzymes and other chemicals. These substances are
released upon rupture of lysosome membrane in inflamma-
tory process that causes tissue damage (Bag et al. 2013).
Lysosomal membrane stabilization is helpful to prevent

13
 M. H. Mahnashi et al.

Fig. 7  Effect of venlafaxine on
mRNA expression of a TNF-α,
a b
b IL-1β, c IL-6, d COX-2 in
CFA-induced arthritis. Results
are expressed as Mean ± SEM
(n = 6) # # p < 0.01, # # #
p < 0.001 compared with
normal control and *p < 0.05,
**p < 0.01, ***p < 0.001
compared with arthritic control
analyzed by one-way ANOVA
followed by Dunnett’s test

c d

release of lysosomal contents like phospholipase A2, pro-
teases and bactericidal enzymes (Shilpa et al. 2018). Studies
have established that non-steroidal anti-inflammatory drugs
stabilize lysosomal membrane by preventing its rupture and
release of contents from lysosome. Because membrane of
red blood cells (RBCs) resemble with lysosomal membrane
in terms of integrity, therefore, RBCs are used in lysosomal
membrane stability study. When erythrocytes are subjected
to hypotonic stress or heat their membrane ruptures and
hemoglobin is released (Bag et al. 2013). Present research
work utilized human red blood cells (HRBCs) to investi-
gate membrane-stabilizing property of venlafaxine. Results
indicated that incubation of venlafaxine reduced the HRBC
membrane rupture and hemolysis (Fig. 1) and has strong
membrane-stabilizing potential as well as anti-inflammatory
activity.
Formaldehyde-induced arthritis is a commonly used acute
model to study anti-inflammatory and anti-arthritic effects
of compounds. Formaldehyde injection into animals paw
causes local pain and inflammation, denaturation of protein
at injection site evokes immunological response (Kumar
et al. 2013; Marius et al. 2018; Uttra and Alamgeer 2017).
The development of arthritis by formaldehyde is biphasic
including neurogenic phase and inflammatory phase. The
first (neurogenic) phase is a result of direct nociceptor stimu-
lation mediated by substance P and bradykinin. Neurogenic
phase measures centrally mediated effects and is insensitive
to anti-inflammatory agents. The second tissue mediated
response (inflammatory phase) is due to changes in cen-
tral procession and release of chemical mediators includ-
ing histamine, serotonin, nitric oxide, prostaglandins and
bradykinin from damaged cells which are responsible for
edema formation and pain stimulation (Anyasor et al. 2014;
Marius et al. 2018; Osman et al. 2017). It has been demon-
strated that drugs having action on CNS impede both phases
uniformly while peripherally acting drugs only inhibit late
phase (Alamgeer et al. 2017a). In the present study, venla-
faxine at oral doses of 25, 50, and 100 mg/kg significantly
reduced the paw edema provoked by injection of formalde-
hyde in sub planter region of rat paw on days 6, 8 and 10
(Table 1). Previously, Aricioglu et al. reported a significant
reduction in paw thickness by intraperitoneal injection of

13
Venlafaxine demonstrated anti‑arthritic activity possibly through down regulation of TNF‑α,…
venlafaxine 50 and 100 mg/kg doses in carrageenan-induced
paw edema model in rats (Aricioğlu et al. 2005). Similarly,
Chugh et al. showed that intraperitoneal administration of
venlafaxine 40 mg/kg significantly reduced the paw inflam-
mation induced by carrageenan (Chugh et al. 2013). Another
study conducted by Ganesh et al. concluded that venlafax-
ine 50 mg/kg had a notable paw volume inhibitory effect
in carrageenan-induced inflammation (Ganesh et al. 2017).
Inline with former reported results our study also depicted
a significant anti-inflammatory effect of venlafaxine 25, 50,
and 100 mg/kg doses. Protein denaturation inhibition and
decrease in inflammatory mediator release might be the
cause of anti-inflammatory effect of venlafaxine.
CFA-induced arthritis is an extensively used preclinical
experimental model for evaluation of anti-arthritic activ-
ity of therapeutic agents. This model has pathological and
clinical similarities to human rheumatoid arthritis includ-
ing joint injury, paw swelling and ankyloses (Anyasor et al.
2014; Jalalpure et al. 2011; Wang et al. 2016). CFA contain
Mycobacterium tuberculosis heat-killed) emulsified in min-
eral oil which activate cell-mediated immune response. CFA
administration causes development of swelling in multiple
joints with inflammatory cells infiltration as well as bone
and cartilage destruction (Hassan et al. 2019; Ingawale and
Patel 2018).
Paw edema and visual arthritis index is employed to
assess the severity of arthritis. The determination of paw
edema and arthritis index is a quick, simple, and sensitive
procedure for assessing the degree of inflammation and ther-
apeutic effects of drugs (Wang et al. 2016). In the current
study CFA administration into rats paw produced edema and
inflammation of non-injected sites. Treatment with venlafax-
ine caused a notable reduction in the paw edema (Fig. 2) and
arthritic index (Fig. 3). These findings showed anti-arthritic
potential of venlafaxine.
Previous studies found that weight loss is more com-
mon in people having RA due to poor appetite and cytokine
production that leads to an increase in protein breakdown
and elevation of resting metabolic rate (Gohil et al. 2018;
Mbiantcha et al. 2017). During inflammation, a decrease in
body weight occurs because of impaired nutrients absorption
through intestine. In a study, it is found that 14C-glucose
and 14C-leucine absorption in rat’s intestine become limited
and anti-inflammatory drugs improve the absorption pro-
cess during inflammatory disease (Alamgeer et al. 2015;
Jalalpure et al. 2011; Patil et al. 2011). In the present study,
rats in arthritic control group significant weight loss. Oral
administration of venlafaxine lead to only slight weight
revival on 28 day (Table 3), it might be due to improvement
in absorption of nutrients through intestine.
During early stage of arthritis increase in paw volume,
hyperalgesia due to prostaglandins, functional impairment
of joints, and lack of mobility were observed in arthritic rats
(Calvino et al. 1987; Nagakura et al. 2003; Schaible et al.
2002). In later stage of disease (day 12 +), adjuvant-induced
arthritic rats become relatively immobile because of sever
paws swelling (Amresh et al. 2007). In the present study,
flexion pain test was used for assessment of hyperalgesia,
and functional impairment was assessed by mobility test and
stance test. Rats in arthritic group had higher flexion pain
and mobility scores while decreased stance score. Treatment
of rats with venlafaxine, significantly reduced the flexion
pain score (Fig. 4a) might be due to its inhibitory effect
on prostaglandins (Kumar et al. 2006). Further, functional
impairment was decreased observed by low mobility score
(Fig. 4b) and high stance score (Fig. 4c) by treatment with
venlafaxine might be due to reduction in paw volume.
Anemia is also associated with rheumatoid arthritis indi-
cated by decreased RBC count and Hb level. Abnormal iron
entrapment in reticuloendothelial system and synovial tissue
causes reduced iron level in plasma that leads to anemia with
failure of bone marrow to combat. Level of plasma iron is
also decreased by IL-1 in alliance with acute phase response
(Ekambaram et al. 2010; Hasan et al. 2018). In the present
study venlafaxine significantly enhanced the RBCs count
and Hb level compared to arthritic control (Table 3) which
illustrates its anti-anemic effect in arthritis. IL-1 suppression
(Fig. 7b) by venlafaxine that in turn improves iron status in
plasma might be accredited for this anti-anemic effect.
There was a remarkable rise in leukocytes and platelets
count in CFA-induced arthritic rats might be due to release
of IL-1 and TNF α (Alamgeer et al. 2017b). The rise in
WBC count in arthritic condition is linked with IL-1β-
mediated rise of relevant colony-stimulating factors. In the
current study, marked elevation in ESR of arthritic rats was
observed and this might be due to endogenous formation
of fibrinogen, α/β globulin, showing an active but obscure
disease activity (Ingawale and Patel 2018; Jalalpure et al.
2011). Administration of venlafaxine significantly reduced
the WBCs, platelets count and ESR level (Table 3) and down
regulation of TNF α and IL-1β expression might be respon-
sible for these effects (Fig. 7).
Liver enzymes such as ALT and AST are also considered
as inflammation markers. A higher levels of these enzymes
indicate hepatic damage (Fan et  al. 2017; Ingawale and
Patel 2018). In the present study, arthritic animals had sig-
nificantly elevated serum ALT and AST level while animals
administered with venlafaxine had reduced level of these
enzymes (Table 3).
Rheumatoid factor (RF) is an auto-antibody produced by
the immune cells against Fc region of IgG and present in
about 80% of patients of rheumatoid arthritis and positively
associated with arthritis progression (Alamgeer et al. 2015;
Gohil et al. 2018). C-reactive protein (CRP) is thought to be
a nonspecific inflammatory marker in rheumatoid arthritis
and its level is firmly linked with radiological severity and
13

progression of disease (Hassan et al. 2019; Uttra et al. 2018).
Unusual increase in blood RF and CRP levels are considered
as a strong hallmark of rheumatoid arthritis (Mbiantcha et al.
2017). Results presented in Table 3 depicts that arthritic rats
had higher sRF and CRP levels and venlafaxine treatment sig-
nificantly reduced the serum level on these parameters.
One of the important tools in diagnosis of rheumatoid
arthritis is radiographic analysis of joints. It gives informa-
tion about severity and remission status of disease. During
early stage of ailment swelling of soft tissue occurs, while joint
space narrowing and bony erosions can be found in the final
stages of arthritis (Ekambaram et al. 2010). Radiographs of
adjuvant-induced arthritic rats in current study have shown in
Fig. 5. In arthritic control animals, soft tissue swelling, joint
space narrowing were seen that stipulate bony destruction. The
venlafaxine treated rats had less degree of soft tissue swelling,
joint space narrowing, and bony destruction.
Pictures of histopathological slides of rats joint have shown
in Fig. 6 having intact synovial lining, normal structure of
connective tissue, absence of inflammation and necrosis in
normal rats. While, arthritic control rats had synovial hyper-
plasia, damage to synovial lining, influx of immune cells,
inflammation, and tissue necrosis and venlafaxine treated
rats showed significant protection against these abnormalities.
Several pro-inflammatory cytokines are thought to have
role in RA pathogenesis. These cytokines are released by
activated immune cells and majorly includes TNF-α and
IL-6. But other cytokines like IL-1β, IL-17, and VEGF also
have their contribution in disease progression (Choy 2012).
TNF-α and IL-1β play key role in rheumatoid arthritis by
liberation of metalloproteases (e.g., collagenases, stromely-
sin) and prostaglandins (PGE2) from activated cells (mac-
rophages, fibroblasts, and synovial dendritic cells). Although
fibroblast-like synoviocytes also express IL-6, however most
prominent feature of these cells is the production of huge
amount of MMPs, prostaglandins and leukotrienes (Aletaha
and Smolen 2018; Bartok and Firestein 2010; Mbiantcha
et al. 2017; McInnes and Schett 2011). Therefore, rheuma-
toid arthritis can be treated by subduing these inflamma-
tory mediators resulting in improved quality of life (Hassan
et al. 2019). In present study, CFA-induced arthritic animals
had significantly raised mRNA expression of TNF-α, IL-1β,
IL-6 and COX-2 and administration of venlafaxine notably
reduced the expression of these mediators (Fig. 7) which
expresses its anti-inflammatory and anti-arthritic potential.
Conclusion
On the basis of results obtained in this study, it is concluded
that venlafaxine demonstrated significant anti-arthritic
activity through inhibition of protein denaturation, HRBC
membrane stabilization, and reduction of paw edema in
13
M. H. Mahnashi et al.
both formaldehyde and CFA-induced arthritis. Venlafax-
ine decreased the arthritic score, flexion pain, and mobility
score while improvements were made on stance score, body
weight, radiographic and histopathological changes, hemato-
logical and biochemical parameters. A possible underlying
mechanism involved in this anti-inflammatory activity is the
inhibition of inflammatory mediators such as TNF-α, IL-1β,
IL-6 and COX-2. Therefore, venlafaxine may be useful in the
treatment of rheumatoid arthritis but, more experimental data
on efficacy and precise mechanism of action are required.
Funding None.
Declarations 
Conflicts of interest  The authors declare that they have no conflict of
interest.
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