INDUCTION, ASSESSMENT, AND TREATMENT

OF REHUMATOID ARTHRITIS IN COLLAGEN

INDUCED RAT MODEL

SUBMITTED BY: IMRAN ABBAS (MPHIL-CH17F18),

RABBIA ZAHID (MPHIL-CH16F18), AMNA SAFDAR
(MPHIL-CH21F18), KUSHIF MIR (MPHIL-CH22F18),
SABA WAHEED (MPHIL-CH25F18)

SUBMITTED TO: DR. ASIM RAZA BASRA

CLASS: MPHIL
SEMESTER: 2ND
COURSE TITLE: CURRENT TOPICS IN
BIOCHEMISTRY
COURSE CODE: MP 262
 Table of Contents

1. Introduction ............................................................................................................................. 3
1.1. Epidemiology ....................................................................................................................... 3
1.2. Arthritic Inflammation and its Different Cellular and Soluble Mediators ........................... 4
1.3. Risk Factors for RA ............................................................................................................. 6
1.4. Animal Models for RA - Studying Disease Pathogenesis and Testing Therapeutic
Agents…………. ............................................................................................................................ 6
1.5. Collagen induced arthritis (CIA) ......................................................................................... 8
1.6. Drugs used for Arthritis Therapy and their Limitations ...................................................... 8
1.6.1. DMARDs ...................................................................................................................... 8
1.6.2. Corticosteroids ............................................................................................................ 10
1.6.3. NSAIDs ...................................................................................................................... 10
1.7. Plant Natural Products - an Alternative for Arthritis Therapy........................................... 11
1.8. Nanoparticle-Based Delivery of Plant Natural Products and Other Drugs for Arthritis
Therapy…….. .. ………………………………………………………………………………….12
2. MATERIALS AND METHODS .......................................................................................... 13
2.1. Materials for purification of Type 2 collagen ................................................................ 13
2.2. Method for Purification of Type 2 Collagen .................................................................. 13
2.3. Animals .......................................................................................................................... 14
2.4. Experimental design and induction of arthritis .............................................................. 14
2.5. Induction and Assessment of Arthritis ........................................................................... 15
2.6. Histopathological examination ....................................................................................... 15
2.7. Further testing to prove Arthritis Development ............................................................. 16
2.7.1. Measurement of Anti –Collagen IgG ...................................................................... 16
2.7.2. Determination of C–reactive protein level .............................................................. 16
2.7.3. Biochemical level Parameters ................................................................................. 17
2.7.4. Enzymes –linked immunosorbent assay for PGE2 and 5-LOX.............................. 17
2.7.5. Determination of mRNA expression level of toll-like receptor TNF-𝛼, IL-4, COX-1, and
COX-2……………………………………………………………………………………….17
2.8. Treatment Methodology ................................................................................................. 19
2.8.1. Method 1 ................................................................................................................. 19
2.8.1.1. Collection of plant specimen ............................................................................... 19

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 2.8.1.2. Preparation of extract .......................................................................................... 19
2.8.1.3. Dose administration to rats.................................................................................. 19
2.8.2. Method 2 ................................................................................................................. 20
2.8.2.1. Materials .............................................................................................................. 20
2.8.2.2. Preparation of Aloe Vera leaf gel extract (AGE): ............................................... 20
2.8.2.3. Preparation of Liposomes encapsulating AGE ................................................... 21
2.8.2.4. Delivery of Liposomes to rats for treatment ....................................................... 21
3. Supposed Results and Discussion.......................................................................................... 22
References ..................................................................................................................................... 24

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 1. Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by painful swelling of
the joints, inflammation of the synovial lining of the joints, hyperplasia, and damage to cartilage
and bone which ultimately decreases life expectancy effecting 1% of total world population [1] .
RA is a multifactorial disorder involving both genetic predisposition and also environmental
components [2, 3].
RA is most prevalent worldwide that is affecting approximately 1.3 million people in the US alone
[4]. Women are more affected by RA than men. It was anticipated that people living long are more
likely to induce RA [5]. Despite a lot of advancement in the field of science and medicine it is still
challenging to diagnose, treat and manage the disease. Usually RA patients are characterized by
joint inflammation and autoantibodies that target various modified self-epitopes. RA is caused by
various factors including environmental genetic and immune system. A number of components of
inflammation and immunity play vital role in governing disease including neutrophils, monocytes,
T and B lymphocytes and vascular endothelium [1, 6].

Common signs of RA are swelling and redness of the hands and feet with pain in the affected
areas. Ulnar deviation, Swan neck deformity [7] and also subcutaneous nodules [8] were among
clinical manifestations of untreated severe RA.

The common serum biomarkers for RA are rheumatoid factor (RF) and anti-citrullinated
protein/peptide antibodies (ACPA). ACPA can also be used as prognostic markers for RA
similarly to RF [9]. Another potential biomarker for RA is oncoprotein survivin, also known
biomarker for cancer. In a study, survivin was found in 50.7% of RA patients but only 5.6% in
controls which indicates its high specificity [10]. There is a striking imbalance between the sexes,
with females representing the majority of autoimmune disease cases. The sex ratio is typically 3:1
[11].

1.1. Epidemiology
The prevalence of RA in some regions is unknown due to the lack of vigorous epidemiological
studies. The prevalence differs in sex, ethnicities and different geographical regions. In Western
countries, prevalence of RA ranges from 0.5% to 1.0% in white individuals. The prevalence of RA

3
in the general black population in Democratic Republic of the Congo, is 0.6%. The prevalence of
RA also differs between ethnicities. In Native American populations a high prevalence of 5–6%
has been reported. The adjusted prevalence ratios were 0.45 for women of Hispanic, 0.69 for Asian
women and 1.02 for African-American descent. Finally, differences in geographical regions have
been reported but these studies are limited, a lower prevalence has been reported in southern
Europe than in northern Europe [12].

1.2. Arthritic Inflammation and its Different Cellular and Soluble

Mediators
Normally the mature T cells encounter self-antigens in periphery but their activation is kept under
control through diverse mechanisms which include unresponsiveness due to inadequate interaction
between the peptide-MHC (major histocompatibility complex) complex and the T cell receptor
(TCR), induction of energy in absence of co-stimulation or suppression by T regulatory (Treg)
cells [13]. RA is initiated by an interplay between components of innate and adaptive immune
responses leading to activation of autoreactive T cells specific for arthritogenic self-antigens in
peripheral lymphoid organs [14]. Antigen-presenting cells (APCs), include dendritic cells,
macrophages and also activated B cells, present arthritogenic autoantigens to T cells having
specific TCRs that recognize these autoantigens. Similarly, upregulation of co-stimulatory
molecules expressed by APCs under inflammatory conditions help in activation of these
arthritogenic T cells. The cytokines such as; interleukin-12 (IL-12) and interferon-γ (IFN-γ) for T
helper 1 (Th1), and IL6 and IL-1β for Th17 help in the differentiation of activated T cells into
pathogenic T cell subsets (Th1, Th17) which are the key drivers of RA pathology [15].

4
Figure 1. Immunopathogenesis of experimental autoimmune arthritis. Schematic representation of the key pathways
is shown: The presentation of an autoantigen to autoreactive T cells and their differentiation into major T helper (Th)
cell subsets under the influence of various cytokines; the activation and secretion of pro-inflammatory cytokines by
myeloid cells; the T-B cell collaboration leading to autoantibody production by plasma cells; and the osteoimmune
cross-talk leading to osteoclast differentiation. These intricate pathways regulate autoimmune inflammation of the
synovial joint as shown by arrows (leading to activation/induction) and blunt ends (leading to suppression/inhibition).

The inflammatory arthritis developed in the joints by the migration of activated pathogenic T cells
from peripheral lymphoid tissues to the joint tissue (synovial tissue) that is mediated primarily by
chemotactic process [16].

These T cells initiated joint-destructive activities by secreting cytokines and other mediators. This
cause inflammation which then attracts other cell types as neutrophils, macrophages and
fibroblasts to that local site together with other effector molecules such as cytokines,
prostaglandins, proteolytic enzymes and osteoclastogenic factors which induce joint inflammation
and also damage cartilage and bone. The B cells cause pathogenesis of RA not through presenting
antigen to the T cells but also by production of cytokines and autoantibodies such as RF and ACPA
that is also increasing the inflammation caused by T cells [17].

5
Th17 cells produced receptor activator of nuclear factor kappa-Β ligand (RANKL), which help in
developing osteoclastogenesis. These osteoclasts then cause bone damage through secreting
matrix-degrading enzymes such as matrix metalloproteinases (MMPs) and cathepsin K [11]

1.3. Risk Factors for RA

The factor which results in progression of RA includes genetic factors, lifestyle, sex-hormones and
environmental factors. Environmental factors which play role in progression of this disease
includes vitamin D deficiency smoking, infectious agents exposure to silica, drugs, obesity and
changes in the microbiota but there is lack of vigorous studies which support this claim [18].

1.4. Animal Models for RA - Studying Disease Pathogenesis and

Testing Therapeutic Agents

Various animal models significantly help to simplify the investigation of complex system
involving swelling, bones deformation, autoimmunity, inflammation and immunological
tolerance. To assess insight view of pathogenic processes of RA in humans, different rodent
models serve as essential tools. The animal models of RA which have proved track record of
predictability in humans are collagen type II induced arthritis in mice and in rats, adjuvant induced
arthritis in rats and antigen induced arthritis in several species. On the other side less frequently
used animal models includes streptococcal cell wall induced arthritis and proteoglycan induced
arthritis[19].
Study of clinical observations and their relation to animal models help us in development of novel
therapeutic treatments for RA. Each animal model comprises of different mechanism which
ultimately drives the disease expression and progression [20].

Animal models have significantly contributed to enlighten the process and mediators that
participate in generation of inflammation and results in bone and cartilage deterioration. No animal
model perfectly duplicate human RA but a perfect animal model for RA should have close possible
reproduction resemblance, complex pathogenesis and symptoms similar to human RA, Current
animal models of RA are extremely reproducible and of short lived duration, having reoccurring
style similar to human disease with some differences, which are (1) quicker progression of the

6
disease pattern , distinguish by severe inflammatory reaction and (2) rodents tends to have marked
resorption inclination and bone formation in reaction to acute joint inflammation [21].

Significant criteria for screening a suitable RA animal model include:

 It must be able to predict the effectiveness of therapeutic agents in humans

 Facility of model execution, data reproducibility, reasonable duration of testing
 The animal model and human disease target should be validated.
 Animal model with a pathogenesis and/or pathology similar to human RA [22].

Among all the models the well-known model is the collagen-induced arthritis (CIA) model in
which B and T cells start a response to CII leading to arthritis induction [23]. Adjuvant-induced
arthritis (AA) model is also mostly used [24]. Many researchers also used proteoglycan-induced
arthritis (PGIA) model that is a T cell–dependent and autoantibody/B cell–driven disease [27].
Table 1 lists some of the common Arthritic models.

Animal model for RA

Induced Models Collagen Induced Arthritis
Adjuvant Induced Arthritis
Streptococcal Cell Wall induced arthritis
Pristane induced Arthritis
Antigen Induced Arthritis
Proteoglycan Induced Arthritis
Genetic Models IL-6R
IL-1Ra
K/BxN
SKG
TNF-
α-transgenic

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1.5. Collagen induced arthritis (CIA)
Collagen induced arthritis is the most widely used induction method for arthritis in rats. For disease
manifestation both T and B cell immunity to autologous type II collagen (CII) are necessarily
required. Important sources of collagen are bovine, chick, porcine and human. Response of
collagen induced arthritis varies with type of strain used and injection conditions. Collagen
induced arthritis (CIA) is most extensively studied animal model because it shares many common
pathological and immunological characteristics with the human RA. The characteristic property of
CIA model is production of auto antibodies self and collagen as well as Infringement of tolerance
[6]. In this case immune system is governed against joint antigen type 2. This makes CIA an ideal
in vivo RA model for studies. In CIA Both T helper Th1 and Th17 responses are induced [28]. To
induced arthritis in rat various varieties of cartilage derived proteins can be used including cartilage
oligomeric matrix protein type II collagen and type XI collagen. Type II collagen is most widely
used. An intradermal infusion of type II collagen homogenized in Freund’s adjuvant results in
serious polyarthritis in rats DA and lewis, starting two weeks after immunization [29]. Both fore
and hind limb paw swells, which exists for a couple of weeks, reducing and then reappearing
leading to severe arthritis with extreme events such as bone distortion. The rat model is used in the
late, chronic stages of arthritis to address the effects of compounds.
The vulnerability to arthritis is associated to particular MHC genes. Since there are numerous
class 2 MHC cells in the joints, it has been postulated that these antigen presenting cells (APCs)
connect and activate CD4+ T cells available in the joints and continuous inflammation. In CIA
key cytokines in rat are TNF-a and IL- 1b [30].

1.6. Drugs used for Arthritis Therapy and their Limitations

Commonly used drugs for RA treatment and their limitations are listed below.

1.6.1. DMARDs
Treatment for RA involves medicines that reduce the incidence of RA joint damage. These
medicines are called disease modifying anti arthritic drugs (DMARDs). These drugs help to
maintain joints by obstructing inflammation. Each DMARD works in different manner. Traditional
DMARDs broadly restrict immune system. Targeted DMARDs block specific immune system

8
pathways. The biological drugs are generated by living cells and act on specific cytokines which
are termed as individual immune proteins [51].

Intervention with the inflammatory responses requires DMARDs among which synthetic
DMARDs (that is, small chemical drugs) are notably differentiated from biological DMARDs (that
is, monoclonal antibodies or, less often, receptor constructs). Biological DMARDs target high
specific proteins that are soluble extracellular and cell-membrane-associated [51].

Methotrexate (MTX) is the golden standards of therapy for RA and is a chemical DMARD. It is
thought that MTX can suppress production of pro-inflammatory cytokines [53, 54]. DMARDs
include monoclonal antibodies targeting tumor necrosis factor (TNF)-α (anti-TNF-α) and IL-6
receptor (anti-IL6R) which are inhibiting these cytokines playing major role in promoting RA
pathogenesis. Anti-TNF-α is currently used alone or with other drugs as MTX [59]. About 10–
30% of patients don’t respond to treatment with anti-TNF-α and 23–46% lost responsiveness. Due
to immunosuppressive effects of blocking TNF-α the RA patients are at risk of recurrent infections
[60]. Anti-IL6R is efficacious in suppressing RA However, anti-IL6R has side-effects as anti-
TNF-α [61, 62]. Because disease-modifying anti-rheumatic drugs (DMARDs) work throughout the
body to fight rheumatic arthritis (RA), their powerful action typically does cause some side effects,
commonly:

 Stomach upset. DMARDs sometimes cause nausea, sometimes with vomiting, or diarrhea. Other
medicines can help treat these symptoms, or they often improve as you get used to the drug. If
the symptoms are too uncomfortable to tolerate, your rheumatologist will try a different
medication.
 Liver problems. These are less common than stomach upset. Your doctor will check blood tests
on a regular basis to make sure your liver is not being harmed.
 Blood problems. DMARDs can affect the immune system and raise the risk of infection.
Infection-fighting white blood cells may also be decreased. Low red blood cells (anemia) can
make you tired more easily. A simple blood test by your doctor every so often will make sure
your blood counts are high enough. You should learn about possible side effects of any medicine
you are taking and discuss them with your doctor until you feel comfortable.

Ahead are listed some of the disease modifying antirheumatic drugs for RA.

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1.6.2. Corticosteroids
Corticosteroids are also used for RA therapy for the past several decades. Corticosteroids inhibit
inflammation by binding at the glucocorticoid receptor (GR) known as NR3C1 (nuclear receptor
subfamily 3 group C member 1) causing the transcription of multiple genes inhibiting several
inflammation pathways. The genes including glucocorticoid-induced leucine zipper protein
(GILZ) and MAP kinase phosphatase-1 (MKP-1) by suppressing the nuclear factor kappa light
chain enhancer of activated B cells (NF-κB) [63]. Corticosteroids can also lead to epigenetic
modification of histones results in reduction of inflammation. Corticosteroids cause side effects
which include osteoporosis, peptic ulcer and increased rate of infections [64, 65].

1.6.3. NSAIDs
NSAIDs (nonsteroidal anti-inflammatory drugs) are a type of pain reliever. At prescription doses,
these drugs also curb inflammation. Recently, they are the most widely utilized class of drugs used
in treatment of RA. This category includes Ibuprofen, Aspirin, and Naproxone. Majority of these

10
target and suppress prostaglandins (PGs) through inhibiting the cyclooxygenase (COX) enzymes
[52]. Patients having NSAIDs dose may experience renal, hepatic, and cardiovascular toxicity.
Certain COX-2-selective agents may cause myocardial infarction (heart attack) and stroke [52].

1.7. Plant Natural Products - an Alternative for Arthritis Therapy

As we saw from the above discussion that the drugs used for the treatment of arthritis have some
of their side effects in the body. Many of them are also expensive. Therefore, the future goal is to
move towards the natural products for the treatment of arthritis [66]. Recently, the mechanism of
action of natural products has been emphasized by National Center for Complementary and
Integrative Health (NCCIH)/National Institutes of Health (NIH), USA.

Some of the natural products known to have anti inflammatory effect are; Aloe Vera, Ginger,
Cinnamon, green tea, garlic, black pepper, and willow bark.Natural products are controlling
arthritic inflammation through different pathways like inhibition of effector molecules (e.g., pro-
inflammatory cytokines and chemokines), activation of anti-inflammatory mediators (e.g., IL-4,
IL-10), regulating Th17/Treg balance, and modulation of the osteo-immune cross-talk [70].

Natural products can modulate the Th17/Treg balance by controlling the level of key cytokines
(e.g., IL-1β, IL-6, and TGF-β (transforming growth factor-β)) and many transcription factors such
as STAT3, RORγt (RAR-related orphan receptor gamma), IRF-4 (interferon Regulatory Factor 4),
and Foxp3 (forkhead box P3) [71]. These are acting through certain cytokines (e.g., IL-17). Natural
products can influence the T cell response and also the osteo-immune cross-talk and bone health
[66].

Therefore, natural products can serve as potential therapeutic agents to treat RA either alone or in
combination with certain mainstream anti-arthritic drugs.

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1.8. Nanoparticle-Based Delivery of Plant Natural Products and
Other Drugs for Arthritis Therapy
A major challenge in the treatment of diseases using natural products is their poor absorption. This
results in loss of bioavailability and efficacy, and it requires consumption of a large amount of the
natural product to achieve the required therapeutic effects. This increase in dosage can lead to
unwanted toxicity [72]. Therefore, novel methods for delivering natural products are needed to
improve therapeutic efficacy as well as reduce toxicity. Nanotechnology is an attractive approach
in this regard [73, 74]. Currently, there are many types of nanoparticles that have been approved
for clinical use, and these nanoparticles are being used for therapeutic and diagnostic purposes [75,
76]. Encapsulating the bioactive natural products into nanoparticles can increase there in vivo
stability, extend their circulation time, and allow for their controlled and sustained release [77].
Furthermore, with suitable modifications, nanoparticles can deliver drugs toa particular target site,
including inflamed tissues [78]. For this purpose the surface of the nanoparticles can be modified
with a peptide, an antibody, a protein, or a small molecule to direct them to the desired inflamed
tissue or organ [79, 80]. The most common types of nanoparticles used for drug delivery for
therapeutic purpose are micelles, lipid nanoparticles, liposomes, polymeric nanoparticles and
emulsions [81].

1.9. Importance of Aloe vera

Aloe vera is the oldest medicinal plant ever known and the most applied medicinal plant
worldwide. Preparations of nanocapsule made from the plant A. vera used in cosmetic and
alternative medicine industries regularly make claims regarding the soothing, moisturizing, anti-
inflammatory and healing properties of A. vera. In this method of producing nanocapsules, the
organic phase containing a certain amount of copolymer, A. vera extract, and olive oil was
prepared in an acetone solvent, and the organic solution was then added drop by drop to the
spinning water at a moderate pace at room temperature. The obtained suspension was immediately
dissolved under sonication and then the acetone was removed under vacuum environment. Finally,
the solutions were transferred into a freeze dryer and the final product was a white powder
nanocapsule containing the extract [82].

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 2. MATERIALS AND METHODS
Collagen induced arthritis can be induced in rats, mice and primates which involves immunization
with type two collagen in adjuvant. Pathological changes include synovitis with infiltration of
polymorph nuclear and mononuclear cells, pannus formation, erosion of bone and cartilage, and
fibrosis [31]. Systemic increase in alpha 1 acid glycoprotein ,tumor necrosis factor –alpha
,interleukin (IL)-17,transforming growth factor beta and chemokine (C-C motif ) together with
local IL-alpha/ beta and TGFB ( enrichment and local lymphoid hyperplasia acts as biomarker of
onset of disease .Systemic up regulation of TNF alpha, IL-6, IL-17, IL-18 and transforming
growth factor beta and activates prostaglandins during acute and chronic disease onset [22].
Collagen induced arthritis coincided with systemic leukocytosis and CD4+T cell increase in blood
and spleen which acts as biomarker [45].

2.1. Materials for purification of Type 2 collagen

Powdered cartilage (Herba Diet), 4M guanidine–HCl, 0.05M Tris–HCl, pH 7.5, 0.5M and 0.1M
acetic acid, Sodium chloride, 70% (V/V) formic acid, 0.02M Na2HPO4, Pepsin from porcine gastric
mucosa. All the chemicals of analytical grade were bought from Sigma Aldrich.

2.2. Method for Purification of Type 2 Collagen

The method of purification of Type 2 collagen from cartilage is based on the studies of E. J Miller
and Herbage et al. Cartilage was powdered in a liquid nitrogen freezer mill. If unavailable, cartilage
may be ground to fine powder by using a pestle and mortar placed in bath of dry ice and liquid
nitrogen. Proteoglycans were removed by suspending powdered cartilage in 5ml of 4M guanidine
–HCL in 0.05M Tris-HCL (pH 7.5), for 24 hours at 4oC and centrifuged at 14000g for 1 hour at
same temperature. Supernatant was discarded cartilage pellet was washed with 0.5M acetic acid
to remove guanidine. Centrifuge again at 14000g for 1 hour at 4oC. To solubilize collagen, suspend
cartilage pellet in 20ml of 0.5M acetic acid. Adjust pH of suspension to 2.8 using 70% formic acid
then add 1g of pepsin for every 20g of cartilage and leave it on stirring for 48hours at 4 oC.
Centrifuge at 14000g for 1 hour at 4oC and discard the pellet. To precipitate type 2 collagen from
supernatant, add NaCl gradually with stirring to give a final concentration of 0.89M. Leave to
equilibrate overnight at 4oC then centrifuge again at 14000g for 1 hour at 4 oC. Dissolve pellet in

13
0.1M acetic acid. Then inactivate the residual pepsin by dialyzing against 0.02M Na2PO4 (pH 9.4).
The collagen is Precipitated. Centrifuge the precipitate at 14000g for 1 hour at 4 oC, then dissolve
pellet in 0.1M acetic acid. Dialyze against 0.1M acetic acid and freeze dry. Store collagen at 4 oC
in a desiccator [30].

2.3. Animals

Rats of 6-8 weeks age and either sex, weighing 150-250g, were kept at animal house, Institute of
Chemistry, University of the Punjab, Lahore. Rats were provided water and standard diet. Animal
were kept at 12 hours dark/light cycle and standard condition of humidity (60-70%) and
temperature of (28±2℃). All the animals were acclimated to the environment for period of one
week before the beginning of experiment.

2.4. Experimental design and induction of arthritis

The rats were randomly divided by balloting method into five groups each containing ten rats. The
groups are as follows:

Group-1: Is the vehicle control which is referred to as the positive control for this experiment.
These group of rats are not injected with FCA nor provided with any treatment.

Group-2: Is the negative control for this experiment which is injected with 1% FCA but provided
with no treatment.

Group-3: Is the experimental group which is given 1% FCA dose and later treated with Aloe Vera
dose of 150ul/Kg.

Group-4: Is the second experimental group which is given the same FCA dose and later treated
with Aloe Vera dose of 250ul/kg.

Group-5: Is the protective group in which 200ul/Kg of Aloe Vera was given to the rats for 7 days
before they were injected with 1% FCA.

Group-6: Is the standard group representing FCA induced inflammatory rats treated with 60 µl of
10mg/ml Indomethacin dose equivalent to 10mg/kg.

14
Arthritis was induced in the rats by injecting 0.2 ml FCA (Mycobacterium butyricum suspended
in mineral oil) in intradermal at two or more site at the base of tail using glass syringe or latex free
syringe and 23-gage needle on day 0 in all group 2, 3, and 4. In group 5 FCA was injected on day
7 and treatment was started thereafter. In group 3, 4, and 6 the treatment was started at 8th day
after the induction of FCA. All the doses were selected as mentioned in previous studies on this
topic.

2.5. Induction and Assessment of Arthritis

Arthritis was induced in all groups accept Group 1. For this purpose, Type2 collagen was dissolved
in 4mg/ml in 0.1M acetic acid overnight at 4oC, with vigorous stirring the dissolved collagen could
be stored at -20oC. To produce incomplete Freund adjuvant, grind Tuberculosis with pestle and
mortar to produce fine powder, then suspended it incomplete Freund adjuvant (3mgM.
Tuberculosis /mL of IFA). This should be carried out in a fume hood with a face mask to prevent
inhalation of Tuberculosis powder. Then dissolve type 2 collagen with equal volume of FCA on
ice, using a syringe the emulsion should be strong enough so that is it must not drip out of the
vessel when inverted.

The mice were then Sedated by intraperitoneal injection of 100ul of 10 %(v/v) Hypnorm diluted
in distilled water. Shave the rumps of mice using electric clipper to facilitate the injection. Inject
the prepared emulsion intradermal at two or more site at the base of tail using glass syringe or latex
free syringe and 23-gage needle. The needle becomes blunt easily so it must be changed frequently.
Each rat should receive 0.2 ml of emulsion in total. The emulsion should be shallow enough to be
visible under the skin.

2.6. Histopathological examination

The rats were monitored for the appearance of arthritic symptoms from day 14 onwards after FCA
was injected. The peak time of arthritis onset is around day 30. To compare histological severity,
the ankle joints of rat were removed at day 28, fixed in 10% formalin, and subsequently decalcified
using hydrochloric acid, ethylenediaminetetra acetic acid, sodium tartrate, and potassium sodium
tartrate containing decalcifying solution. Tissues were embedded in paraffin, and stained with
hematoxylin and eosin (H&E) after slicing into 5μm thick sections. Bone erosion, pannus

15
formation, and infiltration of inflammatory cells were examined by blinded histopathologist.
Results were semi-quantified by giving 0, 1, 2, 3, and 4 numbers to normal, minimal, mild,
moderate, and severe changes, respectively.

2.7. Further testing to prove Arthritis Development

Following tests were performed on all six groups and their results compared to determine the
severity of arthritis development and the reduction in inflammation after treatment procedures.

2.7.1. Measurement of Anti –Collagen IgG

Serum level anti – collagen IgG provide a marker of magnitude of humoral response. For the
measurement of Anti-Collagen IgG first a stock solution of type 2 collagen in 0.05M Tris –
HCL/0.2M sodium chloride (pH 7.4) at 1mg/ml was made. This could be stored at -20oC. Then
the Eliza plate was coated with type 2 collagen at 2-5 ug/mL in 0.05M Tris-HCl/0.2M sodium
chloride (pH7.4) overnight at 4℃. This was Blocked for 1 hour at room temperature with 2%BSA.
Incubate the test sera for two hours at room temperature. Level of anti-collagen IgG may vary
enormously between rat, and it is important to serially dilute the sample to ensure that comparison
is made based on the linear portion of titration curve, A suggested starting dilution is 1/100, with
seven three-to fivefold dilution step. Include standard serum sample on each plate. Pooled serum
from collagen immunized rat or affinity purified anti collagen IgG can be used as standard. Wash
6 times with PBS or tween-20, and then detect bound IgG with HRP-conjugated anti-rat IgG, IgG1
or IgG2. Develop with TMB Substrate. Stop the reaction with 4.5N solution of sulfuric acid and
take absorption at 450nm.

2.7.2. Determination of C–reactive protein level

CRP level were measured based on the agglutination methods by using commercial kit. Test relies
on immunological reaction between CRP in the serum and CRP antisera bound to biologically
inert latex particle. Visible agglutination was observed when serum sample contain high level of
CRP reacted with antisera. CRP were semi quantified according to the lab protocol.

16
2.7.3. Biochemical level Parameters

Various biochemical parameter such as AST, ALT, urea and creatinine level were determined in
blood by using autoanalyzer. Various hematological parameter like, WBC, RBC, and platelet
count, along with HB content were also determined by using automated hemocytometer.

2.7.4. Enzymes –linked immunosorbent assay for PGE2 and 5-LOX

PGE2 and 5-LOX level were measured by ELIZA according to kit manufactured protocol. Briefly,
samples were added in pre-coated well of 96 well microliter plate. Then PGE2/5-LOX antibody
was added, followed by streptavidin-horseradish peroxidase. Plate was gently shaken, sealed and
incubated for 1 hour at 37oC. After this the plate was washed three times with washing solution
and chromogen solution A and B were added subsequently. An incubation period of 10 minutes
was allowed at 37oC away from light. After this, stop solution was added and optical density was
measured at 450nm wavelength.

2.7.5. Determination of mRNA expression level of toll-like receptor TNF-𝜶, IL-4, COX-1, and
COX-2

Total mRNA from blood was extracted by Trizol method, while purity and yield were quantified
through nanodrop. cDNA was synthesized by reverse transcription according to kit manufacture
protocol. Briefly, 500ng total RNA was used per reaction and mixed with 0.5ug of 100uM oilgo
dt18 and nuclease free water. Mixture was heated at 65oC for 5 minutes and subsequently chilled
down by placing on ice. Then 5× reaction buffer (20mM MgCl2, 250Mm KCL, 250mM Tris –
HCL (pH 8.3), and 50mM DTT), 10mM dNTP mix, 20 units of Ribolock RNASE inhibitor, and
200 units of reverse transcriptase enzymes were added and mixture was placed at 42℃ for one
hour. Reaction was terminated by heating at 70oC for 5 minutes. Real time (q PCR) was used to
amplify and quantify the product using Bio –Rad system. Briefly, cDNA template was mixed with
gene specific primer, SYBER Green PCR master mixed with ROX (internal Dye and nuclease free
water and placed thermal cycler with 45 cycle of denaturation (95℃), annealing (60℃), and
termination (72℃). Gene of various markers was picked up from Ensemble Genome Browser and
gene specific primers were designed.

17
PCR product were electrophoresed through a 6% polyacrylamide gel and visualized by ethidium
bromide staining. 10ul amplified DNA was digested with 1ul Hae111 restriction enzyme for 1
hour at 37oC. The band is expected to be between 200-260 base pairs.

PCR amplified RNA is subjected to Northern blotting to check the expression of gene as per lab
protocol.

18
2.8. Treatment Methodology

The rats in group 3, 4, 5 and 6 were treated using different doses of Aloe vera. Aloe vera gel was
given to the rats orally. There are two methods to infuse Aloe vera gel into the rats. Method 1 is
based upon simple gel extraction and feeding it directly to the rats and method 2 involves the
formation of nanocapsules containing Aloe vera gel extract which are then fed to the rats to
examine any anti-arthritic activity of Aloe vera.

2.8.1. Method 1

2.8.1.1. Collection of plant specimen

The Aloe vera leaves were collected from botanical garden, University of the Punjab, Lahore.

2.8.1.2. Preparation of extract

Crude gel was collected by peeling out the outer cuticle and cutting out the gel aseptically into
small pieces. The gel was weighed, mixed with distilled water (1:5 w/v) and then homogenized
to create a homogenate. The sample was freshly prepared every time before use. It contained all
the ingredients of the crude gel in the same proportion as it appears in the leaf. To know the dry
weight of the gel (weight without water parts), each piece was dried separately in an air oven at
37°C for 48 hours and was then weighed. Crude gel was collected by peeling out the outer cuticle
and cutting out the gel aseptically into small pieces. The gel was weighed, mixed with distilled
water (1:5 w/v) and then homogenized to create a homogenate. The sample was freshly prepared
every time before use. It contained all the ingredients of the crude gel in the same proportion as it
appears in the leaf.
2.8.1.3. Dose administration to rats
Treatment was started when the disease was at its’s peak at 21st day. The rats in group 3 were
given 150ul/Kg of the prepared Aloe vera extract. The rats in group 4 were given 250ul/kg of the
Aloe Vera extract and the protective group (group 5) was given 200ul/Kg of the Aloe vera extract
daily for 7 days prior to the day of FCA injection. Standard group (group 6) representing FCA
induced inflammatory rats treated with 60 µl of 10mg/ml Indomethacin dose equivalent to
10mg/kg. Regular dose of Aloe vera was optimized and drug healing power was checked based on
the results of biochemically tested parameter and physical signs and symptoms of the disease. for

19
measuring the percentage inhibition of different doses of Aloe vera gel and the standard we
measured the degree of paw circumference of the rats in millimeters using Vernier caliper after
10, 15, 20, 25, days of FCA injection [83]. Percentage of inhibition (PI%) was measured by the
following formula:

PI% = (Vt -Vo) negative control- (Vt -Vo) treatment group/ (Vt -Vo) negative control X 100

Where,

Vt = final reading of paw circumference,

V0 = Initial reading of paw circumference

2.8.2. Method 2

2.8.2.1. Materials
A soybean lecithin (SLP-WHITE) was purchased from Royal food Pvt Ltd and used without
further purification. The composition of SLP-WHITE was as follows: phosphatidylcholine (20
wt%), phosphatidylethanolamine (26 wt%), phosphatidylinositol (16 wt%), phosphatidic acid (16
wt%), others (22 wt%). A hydrophilic fluorescence probe, 3, 3’-Bis [N,N-bis (carboxymethyl)-
aminomethyl] fluorescein (calcein) (purity 98.0%) was ordered from Cayman chemical company
and used for evaluating the trapping efficiency of liposomes. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution are used for cell
proliferation assay. L-Ascorbic acid was purchased from Ethical laboratories Pvt limited.

2.8.2.2. Preparation of Aloe Vera leaf gel extract (AGE):

Aloe vera leaf was kindly supplied by local companies. The flesh, colorless gel fillet (2.46 kg) was
separated from the green rind, then homogenized in a blender at 80˚C for 30 min. The gel was
frozen at -25˚C, and then left at room temperature to thaw completely. The resulting gel was
centrifuged at 8,000 rpm for 30 min, and then 2.30 kg of the clear yellow supernatant was obtained.
The supernatant was diluted to an appropriate concentration and used as Aloe vera leaf gel extract
(AGE) in the following experiments.

20
2.8.2.3. Preparation of Liposomes encapsulating AGE
Liposomes encapsulating AGE were prepared from SLPWHITE according to the Bangham
method as follows. The lecithin was dissolved with chloroform (2 mL) in a test tube. The solvent
was then removed under the stream of nitrogen gas, and the residual solvent was further dried
overnight under vacuum. The dried lipid films were hydrated with 10 mM Tris-HCl buffer (pH
7.3) containing calcein (0.1 mM) and AGE, and warmed (55-60˚C) above its phase transition
temperature for 10 min. The test tube was then shaken on a vortex mixer for 5 min. The total
lecithin concentration was taken 1wt% and AGE concentration was 0.5wt%. The trapping
efficiency for calcein was determined by a fluorescence method with a spectrofluorometer using
excitation and emission wavelengths at 490 and 520 nm, respectively. The liposomal solution was
diluted 50 times by the Tris-HCl buffer, and the total fluorescence intensity was measured (ITotal).
The calcein presented in the outer aqueous phase was then quenched by complexation with Co2+
(CoCl2, 10 mM), and the fluorescence intensity was measured again (IIn).

Finally, the liposomes were destroyed by adding Triton X-100, and the fluorescence intensity was
measured again. The trapping efficiency was calculated according the Equation 1:

Trapping efficiency (%) = (IIn - ITX ・ r) / (ITotal - ITX ・ r) X 100 (1)

where r is the volume correction factor.

The efficiency was expressed in terms of the average of the values measured at least three times
independently [84].

2.8.2.4. Delivery of Liposomes to rats for treatment

The liposomes encapsulating AGE were fed orally to the rats in group 3, 4, and 5 at day 21. The
rats in group 3 were given 150ul/Kg of the prepared Aloe vera extract encapsulated in liposomes.
The rats in group 4 were given 250ul/kg of the Aloe vera extract encapsulated in liposomes and
the protective group (group 5) was given 200ul/Kg of the Aloe vera extract encapsulated in
liposomes daily for 7 days prior to the day of FCA injection. Standard group (group 6) representing
FCA induced inflammatory rats treated with 60 µl of 10mg/ml Indomethacin dose equivalent to
10mg/kg. Regular dose of Aloe vera was optimized and drug healing power was checked based on
the results of biochemically tested parameter and physical signs and symptoms of the disease. for

21
measuring the percentage inhibition of different doses of Aloe vera gel and the standard we
measured the degree of paw circumference of the rats in millimeters using Vernier caliper after
10, 15, 20, 25, days of FCA injection [83]. Percentage of inhibition (PI%) was measured by the
following formula:

PI% = (Vt -Vo) negative control- (Vt -Vo) treatment group/ (Vt -Vo) negative control X 100

Where,

Vt = final reading of paw circumference,

V0 = Initial reading of paw circumference

3. Expected Results and Discussion

A significant increase in paw circumference would be expected in Group 2,3,4,5,6 [45]. In group
1 there would be no arthritic symptoms at all and group 5 which is the protective group may
experience very few or no arthritic symptoms as it is the protective group. After treatment with
Aloe Vera gel a decrease in paw circumference would be observed in group 3 and 4 with group 4
exhibiting better results as compared to group 3 [23]. If the results after performing the experiment
come as expected it would be signifying the curative and protective properties of Aloe Vera gel
towards arthritic treatment. Histological observations are expected to be likely to those found in
the work of Morgan [45]. Results would show less affected cartilage and joint in the treatment
groups when compared to negative control. Cellular infiltration and cartilage damage would be
expected to be reduced in all the treatment groups. Red blood cells count, white blood cells count
and C-reactive protein IgG levels are also expected to decreased in negative control which shows
the anti-arthritic activity of Aloe Vera gel. More over the intensity of bands in northern blotting
for expression of mRNA of biomarkers may also decrease which would further confirm the healing
properties of Aloe Vera. The protective group is expected to show more or less normal levels of
all the parameter in the experimental time point (day 21 and day 28). There would be no significant
change in the blood sugar level in both arthritic and non-arthritic rats however the arthritic rats
would show significant decrease in total protein level which would be restored upon treatment
with Aloe Vera [83]. Arthritic rats would also, show an increase in serum creatinine level.

22
However, the value returns to normal in the treatment group. Protective group may also show a
decreased in serum creatinine level as compared to negative control. A significant increase in the
mRNA levels of COX-1 and COX-2 would be observed in negative control group as compared to
the vehicle control group. In the experimental control groups the mRNA levels of COX-1 and
COX-2 levels are expected to decrease when provided with adequate treatment. The treatment
results by method 2 might be better than method 1 because of the better absorption, increased
resistance time in the body, sustained drug release, and high surface area to volume ratio [84].

23
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